Truncated activin type ii receptor and methods of use

ABSTRACT

The present invention provides a substantially purified growth differentiation factor (GDF) receptor, including a GDF-8 (myostatin) receptor, as well as functional peptide portions thereof. In addition, the invention provides a virtual representation of a GDF receptor or a functional peptide portion thereof. The present invention also provides a method of modulating an effect of myostatin on a cell by contacting the cell with an agent that affects myostatin signal transduction in the cell. In addition, the invention provides a method of ameliorating the severity of a pathologic condition, which is characterized, at least in part, by an abnormal amount, development or metabolic activity of muscle or adipose tissue in a subject, by modulating myostatin signal transduction in a muscle cell or an adipose tissue cell in the subject. The invention also provides a method of modulating the growth of muscle tissue or adipose tissue in a eukaryotic organism by administering an agent that affects myostatin signal transduction to the organism.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a continuation application of U.S. application Ser.No. 12/360,093 filed Jan. 26, 2009, now pending, which is a continuationapplication of U.S. application Ser. No. 11,051,267 filed Feb. 3, 2005,now pending; which is a continuation application of U.S. applicationSer. No. 09/841,730 filed Apr. 24, 2001, now issued as U.S. Pat. No.6,891,082. The disclosure of each of the prior applications isconsidered part of and is incorporated by reference in the disclosure ofthis application.

GRANT INFORMATION

This invention was made in part with government support under Grant No.RO1HD35887 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to growth differentiation factor (GDF)receptors, and more specifically to GDF-8 (myostatin) receptors, tocompositions that affect myostatin signal transduction in a cell, and tomethods of using such compositions to modulate myostatin signaltransduction in a cell.

2. Background Information

The amount of time, effort and money spent in the United States eachyear by individuals intent on losing weight is staggering. For many ofthese individuals, the goal is not merely to look better, but moreimportantly to avoid the inevitable medical problems associated withbeing overweight.

Greater than half of the adult population in the United States isconsidered to be overweight. Furthermore, twenty to thirty percent ofadult men and thirty to forty percent of adult women in the UnitedStates are considered obese, with the highest rates occurring among thepoor and minorities. Obesity, which is defined a being at least abouttwenty percent above the mean level of adiposity, has dramaticallyincreased in prevalence over the past few decades and is becoming amajor problem among the pediatric population. Twenty percent of allchildren are now considered overweight, a number that represents adoubling over the past five years.

Obesity and the medical problems directly attributable to it are a majorcause of morbidity and mortality throughout the world. Obesity is amajor risk factor for the development of various pathologic conditions,including atherosclerosis, hypertension, heart attack, type II diabetes,gallbladder disease, and certain cancers, and contributes to prematuredeath. Heart disease is the leading cause of mortality in the UnitedStates, and type II diabetes afflicts over 16 million people in theUnited States and is one of the leading causes of death by disease.

More than eighty percent of type II diabetes occurs in obese persons.Although type II diabetes affects all races, it is particularlyprevalent among Native Americans, African Americans and Hispanics.Significantly, type II diabetes, which used to occur almost exclusivelyin adults over age forty, now occurs in children, with reported caseshaving almost tripled over the last five years. Type II diabetes, alsocalled non-insulin dependent diabetes, is characterized by reducedsecretion of insulin in response to glucose and by resistance of thebody to the action of insulin, even though insulin levels in thecirculation generally are normal or elevated. Type II diabetes affectsthe function of a variety of different tissues and organs and can leadto vascular disease, renal failure, retinopathy and neuropathy.

In contrast to the medical problems associated with obesity, the severeweight loss that commonly occurs in patients with certain chronicdiseases also presents a challenge to medical intervention. Themolecular basis for this weight loss, referred to as cachexia, is notwell understood. It is clear, however, that cachexia complicatesmanagement of such diseases and is associated with a poor prognosis forthe patients. The effects of cachexia are evident in the wastingsyndrome that occurs in cancer and AIDS patients.

Although great efforts have been made in attempting to elucidate thebiological processes involved in regulating body weight, the resultshave provided more fanfare than actual value. For example, the discoveryof leptin has been hailed as a breakthrough in understanding themolecular basis for fat accumulation in humans, and, with it, thepromise of a cure for obesity. Studies in animals indicated that leptinis involved in transmitting internal signals regulating appetite, andsuggested leptin could be useful for treating humans suffering fromobesity. Progress in using leptin for treating obesity has been slow,however, and, thus far, leptin has not met initial expectations.

Treatment of the morbidly obese currently is limited to surgery toremove portions of the intestine, thereby reducing the amount of food(and calories) absorbed. For the moderately obese, the only “treatment”is eating a healthy diet and exercising regularly, a method that hasproved modestly successful at best. Thus, a need exists to identify thebiological factors involved in regulating body weight, including muscledevelopment and fat accumulation, such that methods for treatingdisorders such as obesity and cachexia can be developed. The presentinvention satisfies this need and provides additional advantages.

SUMMARY OF THE INVENTION

The present invention relates to a substantially purified GDF receptor.A GDF receptor of the invention can be, for example, a myostatinreceptor, a GDF-11 receptor, or other GDF receptor. A myostatinreceptor, for example, interacts specifically at least with myostatin,and also can interact specifically with one or a few additional matureGDF peptides as well. Polynucleotides encoding a GDF receptor,antibodies that specifically interact with a GDF receptor, and the likealso are provided.

The present invention also relates to a method of modulating an effectof a GDF by affecting signal transduction effected by the GDF. By way ofexample, a method of modulating an effect of myostatin on a cell bycontacting the cell with an agent that affects myostatin signaltransduction in the cell is provided. In one embodiment, the agentalters a specific interaction of myostatin with a myostatin receptorexpressed by the cell, thereby modulating myostatin signal transductionin the cell. The myostatin receptor can be an activin receptor, or canbe any other receptor that can be contacted by a mature myostatin orfunctional peptide portion thereof such that myostatin signaltransduction is activated. In another embodiment, the agent binds to amyostatin receptor, thereby enhancing myostatin binding to the receptoror competing with myostatin for the receptor. As such, the agent canincrease myostatin signal transduction, or can reduce or inhibitmyostatin signal transduction. In still another embodiment, the agentacts intracellularly to alter myostatin signal transduction in the cell.

An agent useful for modulating GDF signal transduction in a cell can bea peptide, a peptidomimetic, a polynucleotide, a small organic molecule,or any other agent, and can act as an agonist of GDF signal transductionor as an antagonist of GDF signal transduction. In one embodiment, thepeptide agent alters a specific interaction of myostatin with amyostatin receptor. Such a peptide agent can be, for example, a peptidethat binds or otherwise sequesters myostatin, thereby affecting theability of myostatin to interact specifically with its receptor. Suchagents are exemplified by a mutant myostatin receptor, for example, asoluble extracellular domain of a myostatin receptor, which canspecifically interact with myostatin; by a myostatin prodomain, whichcan specifically interact with myostatin; and by a mutant myostatinpolypeptide that is resistant to proteolytic cleavage into a prodomainand mature myostatin and can interact specifically with myostatin, andare useful as myostatin signal transduction antagonists, which reduce orinhibit myostatin signal transduction in a cell.

In another embodiment, the peptide agent can specifically interact witha myostatin receptor expressed by a cell, thereby competing withmyostatin for the receptor. Such a peptide agent is exemplified by ananti-myostatin receptor antibody or by an anti-idiotypic antibody of ananti-myostatin antibody. Such a peptide agent provides the additionaladvantage that it can be selected not only for its ability to interactspecifically with a myostatin receptor, thereby competing with myostatinfor the receptor, but can be further selected to have an ability to notactivate or not activate myostatin signal transduction. Thus, a peptideagent that specifically interacts with a myostatin receptor expressed bya cell, and activates myostatin dependent signal transduction can beused as a myostatin agonist to increase myostatin signal transduction inthe cell, whereas a peptide agent that specifically interacts with amyostatin receptor expressed by a cell, but does not activate myostatinsignal transduction can be used as a myostatin antagonist to reduce orinhibit myostatin signal transduction in the cell.

An agent useful in a method of the invention also can be apolynucleotide. Generally, but not necessarily, the polynucleotide isintroduced into the cell, where it effects its function either directly,or following transcription or translation or both. For example, thepolynucleotide agent can encode a peptide, which is expressed in thecell and modulates myostatin activity. Such an expressed peptide can be,for example, a mutant myostatin receptor such as a soluble myostatinreceptor extracellular domain; a myostatin receptor extracellular domainoperatively associated with a membrane anchoring domain; or a mutantmyostatin receptor lacking protein kinase activity.

A peptide expressed from a polynucleotide agent also can be a peptidethat affects the level or activity of an intracellular polypeptidecomponent of a GDF signal transduction pathway. The intracellularpolypeptide can be, for example, an Smad polypeptide such as a dominantnegative Smad, which, as disclosed herein, can affect myostatin signaltransduction in a cell. Thus, a polynucleotide agent can encode adominant negative Smad 2, Smad 3 or Smad 4 polypeptide, which, uponexpression in the cell, reduces or inhibits myostatin signaltransduction in the cell; or can encode a Smad 6 or Smad 7 polypeptide,which, upon expression, decreases myostatin signal transduction in thecell. A polynucleotide agent also can encode an intracellular c-skipolypeptide, the expression of which can reduce or inhibit myostatinsignal transduction.

A polynucleotide agent useful in a method of the invention also can be,or can encode, an antisense molecule, a ribozyme or a triplexing agent.For example, the polynucleotide can be (or can encode) an antisensenucleotide sequence such as an antisense c-ski nucleotide sequence,which can increase myostatin signal transduction in a cell; or anantisense Smad nucleotide sequence, which can increase myostatin signaltransduction or can reduce or inhibit myostatin signal transduction,depending on the particular Smad antisense nucleotide sequence.

The present invention also relates to a method of ameliorating theseverity of a pathologic condition, which is characterized, at least inpart, by an abnormal amount, development or metabolic activity of muscleor adipose tissue in a subject. Such a method encompasses modulating GDFsignal transduction in a cell associated with the pathologic condition,for example, modulating myostatin signal transduction in a muscle cellor an adipose tissue cell in the subject. Various pathologic conditionsare amenable to amelioration using a method of the invention, including,for example, wasting disorders such as cachexia, anorexia, musculardystrophies, neuromuscular diseases; and metabolic disorders such asobesity and type II diabetes.

The present invention further relates to a method of modulating thegrowth of muscle tissue or adipose tissue in a eukaryotic organism byadministering to the organism an agent that affects signal transductionmediated by a GDF receptor. In one embodiment, a method of modulatingthe growth of muscle tissue or adipose tissue is performed byadministering an agent that affects myostatin signal transduction. Inanother embodiment, the agent affects GDF-11 signal transduction, ormyostatin and GDF-11 signal transduction. The agent can be, for example,an agent alters the specific interaction of myostatin with a myostatinreceptor, an agent that reduces or inhibits the specific interaction ofmyostatin with a myostatin receptor, or any other agent as disclosedherein. The eukaryotic organism can be a vertebrate, for example,mammalian, avian or piscine organism, or can be an invertebrate, forexample, a mollusk such as a shrimp, a scallop, a squid, an octopus, asnail, or a slug.

The present invention also relates to a method of identifying an agentthat specifically interacts with a growth differentiation factor (GDF)receptor. Such a screening assay of the invention can be performed, forexample, by contacting a GDF receptor with a test agent, and determiningthat the test agent specifically interacts with the GDF receptor,thereby identifying an agent that specifically interacts with a GDFreceptor. The GDF receptor can be any GDF receptor, particularly amyostatin receptor, and the agent can be a GDF receptor agonist, whichincreases GDF signal transduction, or a GDF receptor antagonist, whichreduces or inhibits GDF signal transduction. Such a method of theinvention is useful for screening a library of test agents, particularlya combinatorial library of test agents.

The present invention also provides a virtual representation of a GDFreceptor or a functional peptide portion of a GDF receptor, for example,a virtual representation of GDF 8 receptor or GDF-11 receptor. In oneembodiment, the virtual representation includes an agent that interactsspecifically with the GDF receptor. As such, the invention furtherprovides a method of identifying an agent that interacts specificallywith a growth differentiation factor (GDF) receptor or a functionalpeptide portion of a GDF receptor by using a computer system. Forexample, the method can be performed by testing a virtual test agent forthe ability to interact specifically with a virtual GDF receptor orfunctional peptide portion thereof; and detecting a specific interactionof the virtual test agent with the virtual GDF receptor or functionalpeptide portion thereof, thereby identifying an agent that interactsspecifically with a GDF receptor or functional peptide portion thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequences of murine promyostatin (SEQ ID NO:4); rat promyostatin (SEQ ID NO: 6); human promyostatin (SEQ ID NO: 2);baboon promyostatin (SEQ ID NO: 10); bovine promyostatin (SEQ ID NO:12); porcine promyostatin (SEQ ID NO: 14); ovine promyostatin (SEQ IDNO: 16); chicken promyostatin (SEQ ID NO: 8), turkey promyostatin (SEQID NO: 18); and zebrafish promyostatin (SEQ ID NO: 20). Amino acids arenumbered relative to the human promyostatin (SEQ ID NO: 2). Dashed linesindicate gaps introduced to maximize homology. Identical residues amongsequences are shaded.

FIG. 2 shows the amino acid sequences of murine promyostatin (SEQ ID NO:4) and zebrafish promyostatin (SEQ ID NO: 20), and portions of the aminoacid sequences of salmon allele 1 promyostatin (SEQ ID NO: 27; “salmon1”) and salmon allele 2 promyostatin (SEQ ID NO: 29; “salmon 2”). Aminoacid position relative to human promyostatin is indicated to left ofeach row (compare FIG. 1; first amino acid of salmon1 corresponds tohuman promyostatin 218; first amino acid of salmon2 corresponds to humanpromyostatin 239). Dashed lines indicate gaps introduced to maximizehomology. Relative amino acid positions, including gaps, is indicatedalong top of each row. Identical residues among sequences are shaded.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a substantially purified peptide portionof a promyostatin polypeptide. Promyostatin, which previously has beenreferred to as growth differentiation factor-8 (GDF-8), comprises anamino terminal prodomain and a C-terminal mature myostatin peptide (seeU.S. Pat. No. 5,827,733). Myostatin activity is effected by the maturemyostatin peptide following its cleavage from promyostatin. Thus,promyostatin is a precursor polypeptide that is proteolytically cleavedto produce active myostatin. As disclosed herein, the myostatinprodomain can inhibit myostatin activity, GDF-11 activity, or both.

The present invention also provides a substantially purified peptideportion of a pro-GDF-11 polypeptide. Pro-GDF-11, which previously hasbeen referred to generally as GDF-11, comprises an amino terminalprodomain and a C-terminal mature GDF-11 peptide (see InternationalPublication No. WO 98/35019, which is incorporated herein by reference).GDF-11 activity is effected by the mature GDF-11 peptide following itscleavage from pro-GDF-11. Thus, pro-GDF-11, like promyostatin, is aprecursor polypeptide that is proteolytically cleaved to produce activeGDF-11. As disclosed herein, the GDF-11 prodomain can inhibit GDF-11activity, myostatin activity, or both.

Promyostatin and pro-GDF-11 are members of the transforming growthfactor-θ (TGF-θ) superfamily, which consists of multifunctionalpolypeptides that control proliferation, differentiation, and otherfunctions in various cell types. The TGF-θ superfamily, whichencompasses a group of structurally-related proteins that affect a widerange of differentiation processes during embryonic development,includes, for example, Mullerian inhibiting substance (MIS), which isrequired for normal male sex development (Behringer et al., Nature345:167, 1990), Drosophila decapentaplegic (DPP) gene product, which isrequired for dorsal-ventral axis formation and morphogenesis of theimaginal disks (Padgett et al., Nature 325:81-84, 1987), the XenopusVg-1 gene product, which localizes to the vegetal pole of eggs (Weeks etal., Cell 51:861-867, 1987), the activins (Mason et al., Biochem.Biophys. Res. Comm. 135:957-964, 1986), which can induce the formationof mesoderm and anterior structures in Xenopus embryos (Thomsen et al.,Cell 63:485, 1990), and the bone morphogenic proteins (BMPs, osteogenin,OP-1), which can induce de novo cartilage and bone formation (Sampath etal., J. Biol. Chem. 265:13198, 1990). The TGF-θ family members caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, hematopoiesis, and epithelialcell differentiation (Massague, Cell 49:437, 1987; Massague, Ann. Rev.Biochem. 67:753-791, 1998; each of which is incorporated herein byreference).

Many of the TGF-θ family members have regulatory effects (positive ornegative) on other peptide growth factors. In particular, certainmembers of the TGF-θ superfamily have expression patterns or possessactivities that relate to the function of the nervous system. Forexample, the inhibins and activins are expressed in the brain (Meunieret al., Proc. Natl. Acad. Sci., USA 85:247, 1988; Sawchenko et al.,Nature 334:615, 1988), and activin can function as a nerve cell survivalmolecule (Schubert et al., Nature 344:868, 1990). Another family member,growth differentiation factor-1 (GDF-1), is nervous system-specific inits expression pattern (Lee, Proc. Natl. Acad. Sci., USA 88:4250, 1991),and other family members such as Vgr-1 (Lyons et al., Proc. Natl. Acad.Sci., USA 86:4554, 1989; Jones et al., Development 111:531, 1991), OP-1(Ozkaynak et al., J. Biol. Chem. 267:25220, 1992), and BMP-4 (Jones etal., Development 111:531, 1991), are also expressed in the nervoussystem. Because skeletal muscle produces a factor or factors thatpromote the survival of motor neurons (Brown, Trends Neurosci. 7:10,1984), the expression of myostatin (GDF-8) and GDF-11 in muscle suggeststhat myostatin and GDF-11 can be trophic factors for neurons. As such,methods for modulating the activity of myostatin, GDF-11, or both can beuseful for treating neurodegenerative diseases such as amyotrophiclateral sclerosis or muscular dystrophy, or for maintaining cells ortissues in culture prior to transplantation.

The proteins of the TGF-θ family are synthesized as large precursorproteins, which subsequently undergo proteolytic cleavage at a clusterof basic residues approximately 110 to 140 amino acids from theC-terminus, resulting in the formation of a prodomain peptide and aC-terminal mature peptide. The C-terminal mature peptides of the membersof this family of proteins are structurally related, and the differentfamily members can be classified into distinct subgroups based on theextent of their homology. Although the homologies within particularsubgroups range from 70% to 90% amino acid sequence identity, thehomologies between subgroups are significantly lower, generally rangingfrom 20% to 50%. In each case, the active species appears to be adisulfide-linked dimer of C-terminal peptide fragments.

Promyostatin and pro-GDF-11 polypeptides have been identified inmammalian, avian and piscine species, and myostatin is active in variousother species, including vertebrates and invertebrates. During embryonicdevelopment and in adult animals, myostatin, for example, is expressedspecifically by cells in the myogenic lineage (McPherron et al., Nature387:83-90, 1997, which is incorporated herein by reference). Duringearly embryogenesis, myostatin is expressed by cells in the myotomecompartment of developing somites. At later embryonic stages and inadult animals, myostatin is expressed widely in skeletal muscle tissue,although the levels of expression vary considerably from muscle tomuscle. Myostatin expression also is detected in adipose tissue,although at lower levels than in muscle. Similarly, GDF-11 is expressedin skeletal muscle and adipose tissue, as well as in adult thymus,spleen and uterus, and also is expressed in brain at various stages ofdevelopment.

Promyostatin polypeptides from various species share substantialsequence identity, and the amino acid sequences of human, murine, ratand chicken mature myostatin C-terminal sequence are 100% identical (seeFIG. 1). Promyostatin polypeptides are exemplified herein (see FIG. 1)by human promyostatin (SEQ ID NO: 2); murine promyostatin (SEQ ID NO:4); rat promyostatin (SEQ ID NO: 6); baboon promyostatin (SEQ ID NO:10); bovine promyostatin (SEQ ID NO: 12); porcine promyostatin (SEQ IDNO: 14); ovine promyostatin (SEQ ID NO: 16); chicken promyostatin (SEQID NO: 8), turkey promyostatin (SEQ ID NO: 18); and zebrafishpromyostatin (SEQ ID NO: 20). Promyostatin polypeptides also areexemplified herein by a polypeptide comprising the portions of salmonallele 1 (SEQ ID NO: 27; “salmon1”) and of salmon allele 2 (SEQ ID NO:29; “salmon2”; see FIG. 2). Nucleic acid molecules encoding thesepromyostatin polypeptides are disclosed herein as SEQ ID NOS: 1, 3, 5,9, 11, 13, 15, 7, 17, 19, 26 and 28, respectively (see, also, McPherronand Lee, Proc. Natl. Acad. Sci., USA 94:12457, 1997, which isincorporated herein by reference). A pro-GDF-11 polypeptide isexemplified herein by human pro-GDF-11 (SEQ ID NO: 25), which is encodedby SEQ ID NO: 24.

In view of the extensive conservation among promyostatin polypeptides,particularly among species as diverse as humans and fish, it would be aroutine matter to obtain polynucleotides encoding myostatin from anyspecies, including the remainders of the salmon1 and salmon2 sequences,and to identify promyostatin or myostatin expression in any species. Inparticular, the mature myostatin sequence shares significant homology toother members of the TGF-θ superfamily, and myostatin contains most ofthe residues that are highly conserved among the other family membersand in other species. Furthermore, myostatin, like the TGF-θs andinhibins, contains an extra pair of cysteine residues in addition to theseven cysteine residues present in virtually all other family members.Myostatin is most homologous to Vgr-1 (45% sequence identity). Likeother members of the TGF-θ superfamily, myostatin is synthesized as alarger precursor promyostatin polypeptide that is proteolytic cleavedinto an active myostatin peptide.

Polynucleotides encoding promyostatin polypeptides of various organismscan be identified using well known procedures and algorithms based onidentity (or homology) to the disclosed sequences. Homology or identityis often measured using sequence analysis software such as the SequenceAnalysis Software Package of the Genetics Computer Group (University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705). Such software matches similar sequences by assigning degrees ofhomology to various deletions, substitutions and other modifications.The terms “homology” and “identity,” when used herein in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or of nucleotides that are the samewhen compared and aligned for maximum correspondence over a comparisonwindow or designated region as measured using any number of sequencecomparison algorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

The term “comparison window” is used broadly herein to include referenceto a segment of any one of the number of contiguous positions, forexample, about 20 to 600 positions, for example, amino acid ornucleotide position, usually about 50 to about 200 positions, moreusually about 100 to about 150 positions, in which a sequence may becompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. Methods ofalignment of sequence for comparison are well-known in the art. Optimalalignment of sequences for comparison can be conducted, for example, bythe local homology algorithm of Smith and Waterman (Adv. Appl. Math.2:482, 1981), by the homology alignment algorithm of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity methodof Person and Lipman (Proc. Natl. Acad. Sci., USA 85:2444, 1988), eachof which is incorporated herein by reference; by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.); or by manual alignment and visualinspection. Other algorithms for determining homology or identityinclude, for example, in addition to a BLAST program (Basic LocalAlignment Search Tool at the National Center for BiologicalInformation), ALIGN, AMAS (Analysis of Multiply Aligned Sequences), AMPS(Protein Multiple Sequence Alignment), ASSET (Aligned SegmentStatistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (BiologicalSequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher),FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS,LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, Las Vegasalgorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences.

A number of genome databases are available for comparison, including,for example, a substantial portion of the human genome is available aspart of the Human Genome Sequencing Project (J. Roach,http://weber.u.Washington.edu/˜roach/human_genome_progress 2.html). Inaddition, at least twenty-one genomes have been sequenced in theirentirety, including, for example, M. genitalium, M. jannaschii, H.influenzae, E. coli, yeast (S. cerevisiae), and D. melanogaster.Significant progress has also been made in sequencing the genomes ofmodel organism such as mouse, C. elegans, and Arabadopsis sp. Severaldatabases containing genomic information annotated with some functionalinformation are maintained by different organizations, and areaccessible via the internet, for example, http://wwwtigr.org/tdb;http://www.genetics.wisc.edu; http://genome-www.stanford.edu/˜ball;http://hiv-web.lanl.gov; http://www.ncbi.nlm.nih.gov;http://www.ebi.ac.uk; http://Pasteur.fr/other/biology; andhttp://www.genome.wi.mit.edu.

One example of a useful algorithm is BLAST and BLAST 2.0 algorithms,which are described by Altschul et al. (Nucleic Acids Res. 25:3389-3402,1977; J. Mol. Biol. 215:403-410, 1990, each of which is incorporatedherein by reference). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov). This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra, 1977, 1990). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci., USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, for example, Karlin and Altschul,Proc. Natl. Acad. Sci., USA 90:5873, 1993, which is incorporated hereinby reference). One measure of similarity provided by BLAST algorithm isthe smallest sum probability (P(N)), which provides an indication of theprobability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a nucleic acid isconsidered similar to a references sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, more preferably less than about0.01, and most preferably less than about 0.001.

In one embodiment, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”). Inparticular, five specific BLAST programs are used to perform thefollowing task:

(1) BLASTP and BLAST3 compare an amino acid query sequence against aprotein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotidesequence database;

(3) BLASTX compares the six-frame conceptual translation products of aquery nucleotide sequence (both strands) against a protein sequencedatabase;

(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and

(5) TBLASTX compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (i.e., aligned) bymeans of a scoring matrix, many of which are known in the art.Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet etal., Science 256:1443-1445, 1992; Henikoff and Henikoff, Proteins17:49-61, 1993, each of which is incorporated herein by reference). Lesspreferably, the PAM or PAM250 matrices may also be used (Schwartz andDayhoff, eds., “Matrices for Detecting Distance Relationships: Atlas ofProtein Sequence and Structure” (Washington, National BiomedicalResearch Foundation 1978)). BLAST programs are accessible through theU.S. National Library of Medicine, for example, at www.ncbi.nlm.nih.gov.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In someembodiments, the parameters may be the default parameters used by thealgorithms in the absence of instructions from the user.

A polynucleotide encoding a promyostatin can be derived from anyorganism, including, for example, mouse, rat, cow, pig, human, chicken,turkey, zebrafish, salmon, finfish, other aquatic organisms and otherspecies. Examples of aquatic organisms include those belonging to theclass Piscina, such as trout, char, ayu, carp, crucian carp, goldfish,roach, whitebait, eel, conger eel, sardine, flying fish, sea bass, seabream, parrot bass, snapper, mackerel, horse mackerel, tuna, bonito,yellowtail, rockfish, fluke, sole, flounder, blowfish, filefish; thosebelonging to the class Cephalopoda, such as squid, cuttlefish, octopus;those belonging to the class Pelecypoda, such as clams (e.g., hardshell,Manila, Quahog, Surf, Soft-shell); cockles, mussels, periwinkles;scallops (e.g., sea, bay, calloo); conch, snails, sea cucumbers; arkshell; oysters (e.g., C. virginica, Gulf, New Zealand, Pacific); thosebelonging to the class Gastropoda such as turban shell, abalone (e.g.green, pink, red); and those belonging to the class Crustacea such aslobster, including but not limited to Spiny, Rock, and American; prawn;shrimp, including but not limited to M. rosenbergii, P. styllrolls, P.indicus, P. jeponious, P. monodon, P. vannemel, M. ensis, S. melantho,N. norvegious, cold water shrimp; crab, including, but not limited to,Blue, rook, stone, king, queen, snow, brown, dungeness, Jonah, Mangrove,soft-shelled; squilla, krill, langostinos; crayfish/crawfish, including,but not limited, to Blue, Marron, Red Claw, Red Swamp, Soft-shelled,white; Annelida; Chordata, including, but not limited to, reptiles suchas alligators and turtles; Amphibia, including frogs; and Echinodermata,including, but not limited to, sea urchins.

The present invention provides substantially purified peptide portionsof a promyostatin polypeptide and substantially purified peptideportions of a pro-GDF-11 polypeptide. As used herein, reference to a“pro-GDF,” for example, promyostatin or pro-GDF-11, means the fulllength polypeptide, including the amino terminal prodomain and thecarboxy terminal biologically active GDF peptide. In addition, theprodomain includes a signal peptide (leader sequence), which comprisesabout the first 15 to 30 amino acids at the amino terminus of theprodomain. The signal peptide can be cleaved from the full lengthpro-GDF polypeptide, which can be further cleaved at an Arg-Xaa-Xaa-Arg(SEQ ID NO: 21) proteolytic cleavage site.

Reference herein to amino acid residues is made with respect to the fulllength pro-GDF polypeptides as shown in FIGS. 1 and 2 (see, also,Sequence Listing). It should also be recognized that reference is madeherein to particular peptides beginning or ending at “about” aparticular amino acid residue. The term “about” is used in this contextbecause it is recognized that a particular protease can cleave a pro-GDFpolypeptide at or immediately adjacent to a proteolytic cleavagerecognition site, or one or a few amino acids from the recognition site.As such, reference, for example, to a myostatin prodomain having asequence of about amino acid residues 1 to 263 of SEQ ID NO: 4 wouldinclude an amino terminal peptide portion of promyostatin that includesthe signal peptide and has a carboxy terminus ending at amino acidresidue 257 to amino acid residue 269, preferably at amino acid residue260 to amino acid residue 266.

Similarly, the signal peptide can be cleaved at any position from aboutamino acid residue 15 to 30 of a pro-GDF polypeptide, for example, atresidue 15, 20, 25 or 30, without affecting the function, for example,of a remaining prodomain. Thus, for convenience, reference is madegenerally herein to a peptide portion of a pro-GDF polypeptide, fromwhich the signal peptide has been cleaved, as beginning at about aminoacid residue 20. However, it will be recognized that cleavage of thesignal peptide can be at any amino acid position within about the first15 to 30 amino terminal amino acids of a pro-GDF polypeptide. As such,reference, for example, to a myostatin prodomain having a sequence ofabout amino acid residues 20 to 263 of SEQ ID NO: 4 would include apeptide portion of promyostatin that lacks about the first 15 to 30amino acids of promyostatin, comprising the signal peptide, and that hasa carboxy terminus ending at amino acid residue 257 to amino acidresidue 269, preferably at amino acid residue 260 to amino acid residue266.

In general, reference is made herein to a pro-GDF polypeptide or a GDFprodomain as beginning at about amino acid 1. In view of the abovedisclosure, however, it will be recognized that such pro-GDFpolypeptides or GDF prodomains that lack the signal peptide also areencompassed within the present invention. Further in this respect, itshould be recognized that the presence or absence of a signal peptide ina peptide of the invention can influence, for example, the compartmentsof a cell through which a peptide, for example, a myostatin prodomainwill traverse and to which the peptide ultimately will localize,including whether the peptide will be secreted from the cell. Thus, thepresent invention further provides a substantially purified signalpeptide portion of a pro-GDF polypeptide. As disclosed herein, such asignal peptide can be used to target an agent, particularly a peptideagent, to the same cellular compartments as the naturally occurring GDFfrom which the signal peptide is derived.

The term “peptide” or “peptide portion” is used broadly herein to meantwo or more amino acids linked by a peptide bond. The term “fragment” or“proteolytic fragment” also is used herein to refer to a product thatcan be produced by a proteolytic reaction on a polypeptide, i.e., apeptide produced upon cleavage of a peptide bond in the polypeptide.Although the term “proteolytic fragment” is used generally herein torefer to a peptide that can be produced by a proteolytic reaction, itshould be recognized that the fragment need not necessarily be producedby a proteolytic reaction, but also can be produced using methods ofchemical synthesis or methods of recombinant DNA technology, asdiscussed in greater detail below, to produce a synthetic peptide thatis equivalent to a proteolytic fragment. In view of the disclosedhomology of promyostatin with other members of the TGF-θ superfamily, itwill be recognized that a peptide of the invention is characterized, inpart, in that it is not present in previously disclosed members of thissuperfamily. Whether a peptide portion of a promyostatin or pro-GDF-11polypeptide is present in a previously disclosed member of the TGF-θsuperfamily readily can be determined using the computer algorithmsdescribed above.

Generally, a peptide of the invention contains at least about six aminoacids, usually contains about ten amino acids, and can contain fifteenor more amino acids, particularly twenty or more amino acids. It shouldbe recognized that the term “peptide” is not used herein to suggest aparticular size or number of amino acids comprising the molecule, andthat a peptide of the invention can contain up to several amino acidresidues or more. For example, a full length mature C-terminal myostatinpeptide contains more than 100 amino acids and a full length prodomainpeptide can contain more than 260 amino acids.

As used herein, the term “substantially purified” or “substantiallypure” or “isolated” means that the molecule being referred to, forexample, a peptide or a polynucleotide, is in a form that is relativelyfree of proteins, nucleic acids, lipids, carbohydrates or othermaterials with which it is naturally associated. Generally, asubstantially pure peptide, polynucleotide, or other moleculeconstitutes at least twenty percent of a sample, generally constitutesat least about fifty percent of a sample, usually constitutes at leastabout eighty percent of a sample, and particularly constitutes aboutninety percent or ninety-five percent or more of a sample. Adetermination that a peptide or a polynucleotide of the invention issubstantially pure can be made using well known methods, for example, byperforming electrophoresis and identifying the particular molecule as arelatively discrete band. A substantially pure polynucleotide, forexample, can be obtained by cloning the polynucleotide, or by chemicalor enzymatic synthesis. A substantially pure peptide can be obtained,for example, by a method of chemical synthesis, or using methods ofprotein purification, followed by proteolysis and, if desired, furtherpurification by chromatographic or electrophoretic methods.

A peptide of the invention can be identified by comparison to apromyostatin or pro-GDF-11 sequence and determining that the amino acidsequence of the peptide is contained within the promyostatin orpro-GDF-11 polypeptide sequence, respectively. It should be recognized,however, that a peptide of the invention need not be identical to acorresponding amino acid sequence of promyostatin or pro-GDF-11. Thus, apeptide of the invention can correspond to an amino acid sequence of apromyostatin polypeptide, for example, but can vary from a naturallyoccurring sequence, for example, by containing one or more D-amino acidsin place of a corresponding L-amino acid; or by containing one or moreamino acid analogs, for example, an amino acid that has been derivatizedor otherwise modified at its reactive side chain. Similarly, one or morepeptide bonds in the peptide can be modified. In addition, a reactivegroup at the amino terminus or the carboxy terminus or both can bemodified. Such peptides can be modified, for example, to have improvedstability to a protease, an oxidizing agent or other reactive materialthe peptide may encounter in a biological environment, and, therefore,can be particularly useful in performing a method of the invention. Ofcourse, the peptides can be modified to have decreased stability in abiological environment such that the period of time the peptide isactive in the environment is reduced.

The sequence of a peptide of the invention also can be modified incomparison to the corresponding sequence in a promyostatin or pro-GDF-11polypeptide by incorporating a conservative amino acid substitution forone or a few amino acids in the peptide. Conservative amino acidsubstitutions include the replacement of one amino acid residue withanother amino acid residue having relatively the same chemicalcharacteristics, for example, the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, for example,substitution of arginine for lysine; or of glutamic for aspartic acid;or of glutamine for asparagine; or the like. Examples of positions of apromyostatin polypeptide that can be modified are evident fromexamination of FIG. 1, which shows various amino acid differences in themyostatin prodomain and mature myostatin peptide that do notsubstantially affect promyostatin or myostatin activity.

The present invention also provides a substantially purified proteolyticfragment of a growth differentiation factor (GDF) polypeptide (a pro-GDFpolypeptide) or a functional peptide portion thereof. Proteolyticfragments of a pro-GDF polypeptide are exemplified herein by proteolyticfragments of a promyostatin polypeptide and proteolytic fragments of apro-GDF-11 polypeptide. As disclosed herein, a peptide portion of apro-GDF polypeptide that is equivalent to a proteolytic fragment of apro-GDF can be produced by a chemical method or a recombinant DNAmethod. In view of the present disclosure, proteolytic fragments ofother GDF polypeptides readily can be made and used.

In general, peptides corresponding to proteolytic fragments of a pro-GDFpolypeptide are exemplified by a carboxy terminal (C-terminal) matureGDF fragment, which can interact specifically with a GDF receptor andaffect GDF signal transduction, and an amino terminal prodomainfragment, which can include a signal peptide and, as disclosed herein,can interact specifically with a pro-GDF polypeptide or mature GDFpeptide and affect its ability to effect GDF signal transduction. Forexample, proteolytic fragments of a promyostatin polypeptide include aC-terminal mature myostatin peptide, which can interact specificallywith a myostatin receptor and induce myostatin signal transduction; andan amino terminal prodomain fragment, which can interact specificallywith myostatin, thereby reducing or inhibiting the ability of myostatinto induce myostatin signal transduction.

A proteolytic fragment of a pro-GDF polypeptide, or a functional peptideportion thereof, is characterized, in part, by having or affecting anactivity associated with the stimulation or inhibition of GDF signaltransduction. For example, a promyostatin polypeptide or functionalpeptide portion thereof can have myostatin receptor binding activity,myostatin signal transduction stimulatory or inhibitory activity,myostatin binding activity, promyostatin binding activity, or acombination thereof. Thus, the term “functional peptide portion,” whenused herein in reference to a pro-GDF polypeptide, means a peptideportion of the pro-GDF polypeptide that can interact specifically withits receptor and stimulate or inhibit GDF signal transduction; that caninteract specifically with a mature GDF or a pro-GDF; or that exhibitscellular localization activity, i.e., the activity of a signal peptide.It should be recognized that a functional peptide portion of full lengthmature myostatin peptide, for example, need not have the same activityof the mature myostatin, including the ability to stimulate myostatinsignal transduction, since functional peptide portions of the maturepeptide can have, for example, an ability to specifically interact witha myostatin receptor without also having the ability to activate thesignal transduction pathway. Methods for identifying such a functionalpeptide portion of a pro-GDF polypeptide, which can be useful as amyostatin antagonist, are disclosed herein or otherwise known in theart. Thus, in one embodiment, a functional peptide portion of apromyostatin polypeptide can interact specifically with a myostatinreceptor, and can act as an agonist to stimulate myostatin signaltransduction or as an antagonist to reduce or inhibit myostatin signaltransduction.

In another embodiment, a functional peptide portion of a promyostatinpolypeptide can interact specifically with a promyostatin polypeptide orwith a mature myostatin peptide, thereby blocking myostatin signaltransduction. Such a functional peptide portion of promyostatin can act,for example, by preventing cleavage of a promyostatin polypeptide tomature myostatin; by forming a complex with a mature myostatin peptide;or by some other mechanism. Where a peptide-myostatin complex is formed,the complex can block myostatin signal transduction, for example, byreducing or inhibiting the ability of the myostatin to interactspecifically with its receptor, or by binding to the receptor in theform that lacks the ability to induce myostatin signal transduction.

Proteolytic fragments of a pro-GDF polypeptide can be produced bycleavage of the polypeptide at a proteolytic cleavage site having aconsensus amino acid sequence Arg-Xaa-Xaa-Arg (SEQ ID NO: 21). Suchproteolytic recognition sites are exemplified by the Arg-Ser-Arg-Arg(SEQ ID NO: 22) sequence shown as amino acid residues 263 to 266 in SEQID NO: 1 (promyostatin) or amino acid residues 295 to 298 of SEQ ID NO:25 (human pro-GDF-11; see, also, relative positions 267 to 270 of FIG.2), and by the Arg-Ile-Arg-Arg (SEQ ID NO: 23) sequence shown as aminoacid residues 263 to 266 in SEQ ID NO: 20.

In addition to the proteolytic cleavage site for the signal peptide,promyostatin polypeptides, for example, contain two additional potentialproteolytic processing sites (Lys-Arg and Arg-Arg). Cleavage of apromyostatin polypeptide at or near the latter proteolytic processingsite, which is contained within the consensus Arg-Xaa-Xaa-Arg (SEQ IDNO: 21) proteolytic cleavage recognition site (see, for example, aminoacid residues 263 to 266 of SEQ ID NO: 2), generates a biologicallyactive C-terminal mature human myostatin fragment. The exemplified fulllength mature myostatin peptides contain about 103 to about 109 aminoacids and have a predicted molecular weight of approximately 12,400daltons (Da). In addition, myostatin can form dimers, which have anexpected molecular weight of about 23 to 30 kiloDaltons (kDa). Thedimers can be myostatin homodimers or can be heterodimers, for example,with GDF-11 or another GDF or TGF-θ family member.

A proteolytic fragment of the invention is exemplified by a GDFprodomain, for example, a myostatin prodomain, which includes aboutamino acid residues 20 to 262 of a promyostatin polypeptide, or afunctional peptide portion thereof, or a GDF-11 prodomain, whichincludes about amino acid residues 20 to 295 of a pro-GDF-11polypeptide, or a functional peptide portion thereof, each of which canfurther contain the signal peptide comprising about amino acids 1 to 20of the respective pro-GDF polypeptide. Myostatin prodomains are furtherexemplified by about amino acid residues 20 to 263 as set forth in SEQID NO: 4 and SEQ ID NO: 6; as well as by about amino acid residues about20 to 262 as set forth in SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:20, which can be produced by proteolytic cleavage of a correspondingpromyostatin polypeptide, can be chemically synthesized, or can beexpressed from a recombinant polynucleotide encoding the proteolyticfragment. A functional peptide portion of a myostatin prodomain isexemplified by a peptide portion of a myostatin prodomain that caninteract specifically with myostatin or with promyostatin. A GDF-11prodomain is exemplified by about amino acid residues 20 to 295 of SEQID NO: 25, which can further include the signal peptide comprising aboutamino acid residues 1 to 20 of SEQ ID NO: 25, and a functional peptideportion of a GDF-11 prodomain is exemplified by a peptide portion of aGDF-11 prodomain that can specifically interact with mature GDF-11 or apro-GDF-11 polypeptide. Preferably, the functional peptide portion of aGDF prodomain inhibits the ability of the corresponding GDF or a relatedGDF to stimulate signal transduction, for example, by reducing orinhibiting the ability of the GDF to interact specifically with itsreceptor, or by binding to the receptor as an inactive complex. In oneembodiment, the present invention provides a functional fragment of apro-GDF polypeptide, particularly a functional fragment of a GDFprodomain, operably linked to a GDF signal peptide, preferably amyostatin signal peptide or a GDF-11 signal peptide comprising about thefirst 15 to 30 amino terminal amino acids of promyostatin or pro-GDF-11,respectively.

As disclosed herein, a myostatin prodomain or GDF-11 prodomain caninteract with mature myostatin, GDF-11, or both, thereby reducing orinhibiting the ability of the mature GDF to interact specifically withits receptor (see Examples 7 and 8). Thus, a functional peptide portionof a myostatin prodomain, for example, can be obtained by examiningpeptide portions of a myostatin prodomain using methods as providedherein, and identifying functional peptide portions of the prodomainthat can interact specifically with myostatin or with promyostatin andcan reduce or inhibit the ability of myostatin to interact specificallywith a myostatin receptor or to stimulate myostatin signal transduction.

A functional peptide portion of a myostatin prodomain that canspecifically interact with myostatin, or a functional peptide portion ofanother GDF prodomain, also can be identified using any of variousassays known to be useful for identifying specific protein-proteininteractions. Such assays include, for example, methods of gelelectrophoresis, affinity chromatography, the two hybrid system ofFields and Song (Nature 340:245-246, 1989; see, also, U.S. Pat. No.5,283,173; Fearon et al., Proc. Natl. Acad. Sci., USA 89:7958-7962,1992; Chien et al., Proc. Natl. Mad. Sci. USA 88:9578-9582, 1991; Young,Biol. Reprod. 58:302-311 (1998), each of which is incorporated herein byreference), the reverse two hybrid assay (Leanna and Hannink, Nucl.Acids Res. 24:3341-3347, 1996, which is incorporated herein byreference), the repressed transactivator system (U.S. Pat. No.5,885,779, which is incorporated herein by reference), the phage displaysystem (Lowman, Ann. Rev. Biophys. Biomol. Struct. 26:401-424, 1997,which is incorporated herein by reference), GST/HIS pull down assays,mutant operators (WO 98/01879, which is incorporated herein byreference), the protein recruitment system (U.S. Pat. No. 5,776,689,which is incorporated herein by reference), and the like (see, forexample, Mathis, Clin. Chem. 41:139-147, 1995 Lam, Anticancer Drug Res.12:145-167, 1997; Phizicky et al., Microbiol. Rev. 59:94-123, 1995; eachof which is incorporated herein by reference).

A functional peptide portion of a GDF prodomain also can be identifiedusing methods of molecular modeling. For example, an amino acid sequenceof a mature myostatin peptide can be entered into a computer systemhaving appropriate modeling software, and a three dimensionalrepresentation of the myostatin (“virtual myostatin”) can be produced. Apromyostatin amino acid sequence also can be entered into the computersystem, such that the modeling software can simulate portions of thepromyostatin sequence, for example, portions of the prodomain, and canidentify those peptide portions of the prodomain that can interactspecifically with the virtual myostatin. A base line for a specificinteraction can be predefined by modeling the virtual myostatin and afull length promyostatin prodomain, and identifying the amino acidresidues in the virtual myostatin that are “contacted” by the prodomain,since such an interaction is known to inhibit the activity of themyostatin.

It should be recognized that such methods, including two hybrid assaysand molecular modeling methods, also can be used to identify otherspecifically interacting molecules encompassed within the presentinvention. Thus, methods such as the two hybrid assay can be used toidentify a GDF receptor such as a myostatin receptor using, for example,a myostatin peptide or a peptide portion thereof that specificallyinteracts with an Act RIIA or Act RIIB receptor as one binding componentof the assay, and identifying a GDF receptor, which specificallyinteracts with the myostatin peptide. Similarly, methods of molecularmodeling can be used to identify an agent that interacts specificallywith a mature GDF peptide such as mature myostatin, or with a GDFreceptor and, therefore, can be useful as an agonist or an antagonist ofsignal transduction mediated by the GDF or the GDF receptor. Such anagent can be, for example, a functional peptide portion of a myostatinprodomain or GDF-11 prodomain, or a chemical agent that mimics theaction of the GDF prodomain.

Modeling systems useful for the purposes disclosed herein can be basedon structural information obtained, for example, by crystallographicanalysis or nuclear magnetic resonance analysis, or on primary sequenceinformation (see, for example, Dunbrack et al., “Meeting review: theSecond meeting on the Critical Assessment of Techniques for ProteinStructure Prediction (CASP2) (Asilomar, Calif., Dec. 13-16, 1996). FoldDes. 2(2): R27-42, (1997); Fischer and Eisenberg, Protein Sci. 5:947-55,1996; (see, also, U.S. Pat. No. 5,436,850); Havel, Prog. Biophys. Mol.Biol. 56:43-78, 1991; Lichtarge et al., J. Mol. Biol. 274:325-37, 1997;Matsumoto et al., J. Biol. Chem. 270:19524-31, 1995; Sali et al., J.Biol. Chem. 268:9023-34, 1993; Sali, Molec. Med. Today 1:270-7, 1995a;Sali, Curr. Opin. Biotechnol. 6:437-51, 1995b; Sali et al., Proteins 23:318-26, 1995c; Sali, Nature Struct. Biol. 5:1029-1032, 1998; U.S. Pat.No. 5,933,819; U.S. Pat. No. 5,265,030, each of which is incorporatedherein by reference).

The crystal structure coordinates of a promyostatin polypeptide or a GDFreceptor can be used to design compounds that bind to the protein andalter its physical or physiological properties in a variety of ways. Thestructure coordinates of the protein can also be used to computationallyscreen small molecule data bases for agents that bind to the polypeptideto develop modulating or binding agents, which can act as agonists orantagonists of GDF signal transduction. Such agents can be identified bycomputer fitting kinetic data using standard equations (see, forexample, Segel, “Enzyme Kinetics” (J. Wiley & Sons 1975), which isincorporated herein by reference).

Methods of using crystal structure data to design inhibitors or bindingagents are known in the art. For example, GDF receptor coordinates canbe superimposed onto other available coordinates of similar receptors,including receptors having a bound inhibitor, to provide anapproximation of the way the inhibitor interacts with the receptor.Computer programs employed in the practice of rational drug design alsocan be used to identify compounds that reproduce interactioncharacteristics similar to those found, for example, between a maturemyostatin and a co-crystallized myostatin prodomain. Detailed knowledgeof the nature of the specific interactions allows for the modificationof compounds to alter or improve solubility, pharmacokinetics, and thelike, without affecting binding activity.

Computer programs for carrying out the activities necessary to designagents using crystal structure information are well known. Examples ofsuch programs include, Catalyst Databases™—an information retrievalprogram accessing chemical databases such as BioByte Master File,Derwent WDI and ACD; Catalyst/HYPO™—generates models of compounds andhypotheses to explain variations of activity with the structure of drugcandidates; Ludi™-fits molecules into the active site of a protein byidentifying and matching complementary polar and hydrophobic groups; andLeapfrog™—“grows” new ligands using a genetic algorithm with parametersunder the control of the user.

Various general purpose machines can be used with such programs, or itmay be more convenient to construct more specialized apparatus toperform the operations. Generally, the embodiment is implemented in oneor more computer programs executing on programmable systems eachcomprising at least one processor, at least one data storage system(including volatile and non-volatile memory and/or storage elements), atleast one input device, and at least one output device. The program isexecuted on the processor to perform the functions described herein.

Each such program can be implemented in any desired computer language,including, for example, machine, assembly, high level procedural, orobject oriented programming languages, to communicate with a computersystem. In any case, the language may be a compiled or interpretedlanguage. The computer program will typically be stored on a storagemedia or device, for example, a ROM, CD-ROM, magnetic or optical media,or the like, that is readable by a general or special purposeprogrammable computer, for configuring and operating the computer whenthe storage media or device is read by the computer to perform theprocedures described herein. The system may also be considered to beimplemented as a computer-readable storage medium, configured with acomputer program, where the storage medium so configured causes acomputer to operate in a specific and predefined manner to perform thefunctions described herein.

Embodiments of the invention include systems, for example, internetbased systems, particularly computer systems which store and manipulatecoordinate information obtained by crystallographic or NMR analysis, oramino acid or nucleotide sequence information, as disclosed herein. Asused herein, the term “computer system” refers to the hardwarecomponents, software components, and data storage components used toanalyze coordinates or sequences as set forth herein. The computersystem typically includes a processor for processing, accessing andmanipulating the sequence data. The processor can be any well known typeof central processing unit, for example, a Pentium II or Pentium IIIprocessor from Intel Corporation, or a similar processor from Sun,Motorola, Compaq, Advanced MicroDevices or International BusinessMachines.

Typically the computer system is a general purpose system that comprisesthe processor and one or more internal data storage components forstoring data, and one or more data retrieving devices for retrieving thedata stored on the data storage components. A skilled artisan canreadily appreciate that any one of the currently available computersystems are suitable.

In one embodiment, the computer system includes a processor connected toa bus, which is connected to a main memory, preferably implemented asRAM, and one or more internal data storage devices such as a hard driveor other computer readable media having data recorded thereon. In someembodiments, the computer system further includes one or more dataretrieving devices for reading the data stored on the internal datastorage devices.

The data retrieving device may represent, for example, a floppy diskdrive, a compact disk drive, a magnetic tape drive, or a modem capableof connection to a remote data storage system (e.g., via the internet).In some embodiments, the internal data storage device is a removablecomputer readable medium such as a floppy disk, a compact disk, amagnetic tape, etc. containing control logic and/or data recordedthereon. The computer system may advantageously include or be programmedby appropriate software for reading the control logic and/or the datafrom the data storage component once inserted in the data retrievingdevice.

The computer system generally include a display, which is used todisplay output to a computer user. It should also be noted that thecomputer system can be linked to other computer systems in a network orwide area network to provide centralized access to the computer system.

Where it is desired to identify a chemical entity that interactsspecifically with myostatin or with a GDF receptor, any of severalmethods to screen chemical entities or fragments for their ability tointeract specifically with the molecule can be used. This process maybegin by visual inspection, for example, of myostatin and a myostatinprodomain on the computer screen. Selected peptide portions of theprodomain, or chemical entities that can act as mimics, then can bepositioned in a variety of orientations, or docked, within an individualbinding site of the myostatin. Docking can be accomplished usingsoftware such as Quanta and Sybyl, followed by energy minimization andmolecular dynamics with standard molecular mechanics forcefields, suchas CHARMM and AMBER.

Specialized computer programs can be particularly useful for selectingpeptide portions of a prodomain, or chemical entities useful, forexample, as a GDF receptor agonist or antagonist. Such programs include,for example, GRID (Goodford, J. Med. Chem., 28:849-857, 1985; availablefrom Oxford University, Oxford, UK); MCSS (Miranker and Karplus,Proteins: Structure. Function and Genetics 11:29-34, 1991, availablefrom Molecular Simulations, Burlington Mass.); AUTODOCK (Goodsell andOlsen, Proteins: Structure. Function, and Genetics 8:195-202, 1990,available from Scripps Research Institute, La Jolla Calif.); DOCK(Kuntz, et al., J. Mol. Biol. 161:269-288, 1982, available fromUniversity of California, San Francisco Calif.), each of which isincorporated herein by reference.

Suitable peptides or agents that have been selected can be assembledinto a single compound or binding agent. Assembly can be performed byvisual inspection of the relationship of the fragments to each other onthe three-dimensional image displayed on a computer screen, followed bymanual model building using software such as Quanta or Sybyl. Usefulprograms to aid one of skill in the art in connecting the individualchemical entities or fragments include, for example, CAVEAT (Bartlett etal, Special Pub., Royal Chem. Soc. 78:182-196, 1989, available from theUniversity of California, Berkeley Calif.); 3D Database systems such asMACCS-3D (MDL Information Systems, San Leandro Calif.; for review, seeMartin, J. Med. Chem. 35:2145-2154, 1992); HOOK (available fromMolecular Simulations, Burlington, Mass.), each of which is incorporatedherein by reference.

In addition to the method of building or identifying such specificallyinteracting agents in a step-wise fashion, one fragment or chemicalentity at a time as described above, the agents can be designed as awhole or de novo using either an empty active site or, optionally,including some portions of a known agent that specifically interacts,for example, a full length myostatin prodomain, which interactsspecifically with myostatin. Such methods include, for example, LUDI(Bohm, J. Comp. Aid. Molec. Design 6:61-78, 1992, available from BiosymTechnologies, San Diego Calif.); LEGEND (Nishibata and Itai, Tetrahedron47:8985, 1991, available from Molecular Simulations, Burlington Mass.);LeapFrog (available from Tripos Associates, St. Louis Mo.), and thosedescribed by Cohen et al. (J. Med. Chem. 33:883-894, 1990) and by Naviaand Murcko, Curr. Opin. Struct. Biol. 2:202-210, 1992, each of which isincorporated herein by reference).

Specific computer software is available in the art to evaluate compounddeformation energy and electrostatic interaction. Examples of programsdesigned for such uses include Gaussian 92, revision C (Frisch,Gaussian, Inc., Pittsburgh Pa., 1992); AMBER, version 4.0 (Kollman,University of California at San Francisco, 1994); QUANTA/CHARMM(Molecular Simulations, Inc., Burlington Mass., 1994); and InsightII/Discover (Biosysm Technologies Inc., San Diego Calif., 1994). Theseprograms may be implemented using, for example, a Silicon Graphicsworkstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550. Otherhardware systems and software packages will be known to those skilled inthe art of which the speed and capacity are continually modified.

A molecular modeling process for identifying an agent that interactsspecifically with a molecule of interest, for example, with a mature GDFpeptides such as mature myostatin, or with a GDF receptor can beperformed as disclosed herein. In a first step, a virtual representationof a target molecule, for example, myostatin, is performed. Thus, in oneembodiment, the present invention provides a virtual representation of atarget molecule, wherein the target molecule is selected from a pro-GDFpolypeptide, for example, promyostatin; a peptide portion of a pro-GDFpolypeptide; a GDF receptor; and a relevant domain of a GDF receptor,for example, a GDF binding domain. The virtual representation of thetarget molecule can be displayed or can be maintained in a computersystem memory. The process begins at a start state, comprising thevirtual target molecule, then moves to a state wherein a databasecontaining one or more virtual test molecules stored to a memory in thecomputer system. As discussed above, the memory can be any type ofmemory, including RAM or an internal storage device.

The process then moves to a state wherein the ability of a virtual firsttest molecule to specifically interact with the virtual target moleculeis determined, wherein the database containing the virtual testmolecule, which can be one of a population of test molecules, is openedfor analysis of the an interaction of the virtual target molecule andvirtual test molecule, and the analysis is made. A determination of aspecific interaction can be made based on calculations performed bysoftware maintained in the computer system, or by comparison to apredetermined specific interaction, which can be stored in a memory inthe computer system and accessed as appropriate.

The process then moves to a state wherein, where a specific interactionis detected, the virtual test molecule is displayed, or is stored in asecond database on the computer. If appropriate, the process is repeatedfor the virtual target molecule and a second virtual test molecule, athird virtual test molecule, and so on, as desired.

If a determination is made that a virtual test molecule specificallyinteracts with the virtual target molecule, the identified virtual testmolecule is moved from the database and can be displayed to the user.This state notifies the user that the molecule with the displayed nameor structure interacts specifically with the target molecule within theconstraints that were entered. Once the name of the identified testmolecule is displayed to the user, the process moves to a decisionstate, wherein a determination is made whether more virtual testmolecules exist in the database or are to be examined. If no moremolecules exist in the database, then the process terminates at an endstate. However, if more test molecules exist in the database, then theprocess moves to a state, wherein a pointer is moved to the next testmolecule in the database so that it can be examined for specific bindingactivity. In this manner, the new molecule is examined for the abilityto interact specifically with the virtual target molecule.

Such methods as described above can be used in various aspectsencompassed within the claimed invention. Thus, the methods can be usedto identify a peptide portion of a promyostatin prodomain that caninteract specifically with myostatin and reduce or inhibit the abilityof myostatin to interact with its receptor or otherwise affect theability of the myostatin to effect signal transduction. Similarly, themethods can be used to identify small organic molecules that mimic theaction of a GDF prodomain, thereby reducing or inhibiting myostatin orGDF-11 signal transduction. The methods also can be used to identifyagents that interact specifically with a GDF receptor, for example, anAct RIIA, Act RIIB or other GDF receptor, such agents being useful asGDF receptor agonists or antagonists, which can modulate GDF signaltransduction in a cell. In addition, the methods provide a means toidentify previously unknown pro-GDF polypeptides or GDF receptors, forexample, by identifying conserved structural features of the particularpolypeptides.

Similar to other members of the TGF-θ superfamily, active GDF peptidesare expressed as precursor polypeptides, which are cleaved to a mature,biologically active form. Accordingly, in still another embodiment, theproteolytic fragment of a pro-GDF polypeptide is a mature GDF peptide,or a functional peptide portion of a mature GDF peptide, where, asdiscussed above, the functional peptide portion can have the activity ofa GDF agonist or antagonist. The proteolytic fragment can be a matureC-terminal myostatin peptide, which includes about amino acid residues268 to 374 of a promyostatin polypeptide (see FIG. 1; see, also, FIG.2), or a mature C-terminal GDF-11 peptide, which includes about aminoacid residues 299 to 407 of a pro-GDF-11 polypeptide. Full length maturemyostatin peptides are exemplified by amino acid residues about 268 to375 as set forth in SEQ ID NO: 4 and SEQ ID NO: 6; by amino acidresidues about 267 to 374 as set forth in SEQ ID NO: 2, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 14, SEQ ID NO:16, and SEQ ID NO: 20, and by amino acid residues about 49 to 157 of SEQID NO: 27 and amino acid residues about 28 to 136 of SEQ ID NO: 29. Afull length mature GDF-11 peptide is exemplified by amino acid residuesabout 299 to 407 of SEQ ID NO: 25. Functional peptide portions of themature GDF peptides are exemplified by peptide portions of maturemyostatin or mature GDF-11 that have an agonist or antagonist activitywith respect to the activity of a mature GDF peptide. Preferably, themature GDF peptide activity is an ability to interact specifically withits receptor.

As disclosed herein, a mature myostatin peptide (referred to hereingenerally as “myostatin”) can induce myostatin signal transductionactivity by interacting specifically with a myostatin receptor expressedon the surface of a cell (see Example 7). Thus, a functional peptideportion of myostatin can be obtained by examining peptide portions of amature myostatin peptide using a method as described herein (Example 7)or otherwise known in the art, and identifying functional peptideportions of myostatin that specifically interact with a myostatinreceptor, for example, an activin type IIA receptor (Act RIIA) or ActRIIB receptor expressed on a cell (Act RIIA, Cell 65:973-982, 1991; ActRIIB Cell 68:97-108, 1992, both herein incorporated by reference intheir entirety).

A myostatin prodomain can reduce or inhibit myostatin signaltransduction activity. In one embodiment, the myostatin prodomain caninteract specifically with myostatin, thereby reducing or inhibiting theability of the myostatin peptide to interact specifically with itsreceptor. As disclosed herein, a precursor promyostatin also lacks theability to interact specifically with a myostatin receptor, and,therefore, mutations in promyostatin that reduce or inhibit the abilityof promyostatin to be cleaved into mature myostatin provide a means toreduce or inhibit myostatin signal transduction. Accordingly, in anotherembodiment, the present invention provides a mutant pro-GDF polypeptide,which contains one or more amino acid mutations that disrupt proteolyticcleavage of the mutant pro-GDF to an active mature GDF peptide.

A mutant pro-GDF polypeptide of the invention can have a mutation thataffects cleavage at a proteolytic cleavage site such as the consensusproteolytic cleavage recognition site Arg-Xaa-Xaa-Arg (SEQ ID NO: 21),which is present in pro-GDF polypeptides. Thus, the mutation can be amutation of an Arg residue of SEQ ID NO: 21, such that a mutantpromyostatin, for example, cannot be cleaved into a myostatin prodomainand a mature myostatin peptide. However, the mutation also can be at asite other than the proteolytic cleavage site, and can alter the abilityof the protease to bind to the pro-GDF polypeptide so as to effectproteolysis at the cleavage site. A mutant pro-GDF polypeptide of theinvention, for example, a mutant promyostatin or mutant pro-GDF-11 canhave a dominant negative activity with respect to myostatin or GDF-11and, therefore, can be useful for reducing or inhibiting myostatin orGDF-11 signal transduction in a cell.

The present invention also provides a substantially purifiedpolynucleotide, which encodes a peptide portion of a promyostatinpolypeptide or a mutant promyostatin, or a peptide portion of apro-GDF-11 polypeptide or mutant pro-GDF-11, as described above. Asdiscussed in greater detail below, the invention also providespolynucleotides useful as agents for modulating the affect of myostatinon a cell, and further provides a polynucleotide encoding a GDFreceptor, or functional peptide portion thereof. Examples of suchpolynucleotides are provided in the following disclosure. As such, itshould be recognized that the following disclosure is relevant to thevarious embodiments of the invention as disclosed herein.

The term “polynucleotide” is used broadly herein to mean a sequence oftwo or more deoxyribonucleotides or ribonucleotides that are linkedtogether by a phosphodiester bond. As such, the term “polynucleotide”includes RNA and DNA, which can be a gene or a portion thereof, a cDNA,a synthetic polydeoxyribonucleic acid sequence, or the like, and can besingle stranded or double stranded, as well as a DNA/RNA hybrid.Furthermore, the term “polynucleotide” as used herein includes naturallyoccurring nucleic acid molecules, which can be isolated from a cell, aswell as synthetic molecules, which can be prepared, for example, bymethods of chemical synthesis or by enzymatic methods such as by thepolymerase chain reaction (PCR). In various embodiments, apolynucleotide of the invention can contain nucleoside or nucleotideanalogs, or a backbone bond other than a phosphodiester bond (seeabove).

In general, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. However, a polynucleotidealso can contain nucleotide analogs, including non-naturally occurringsynthetic nucleotides or modified naturally occurring nucleotides. Suchnucleotide analogs are well known in the art and commercially available,as are polynucleotides containing such nucleotide analogs (Lin et al.,Nucl. Acids Res. 22:5220-5234 (1994); Jellinek et al., Biochemistry34:11363-11372 (1995); Pagratis et al., Nature Biotechnol. 15:68-73(1997), each of which is incorporated herein by reference).

The covalent bond linking the nucleotides of a polynucleotide generallyis a phosphodiester bond. However, the covalent bond also can be any ofnumerous other bonds, including a thiodiester bond, a phosphorothioatebond, a peptide-like bond or any other bond known to those in the art asuseful for linking nucleotides to produce synthetic polynucleotides(see, for example, Tam et al., Nucl. Acids Res. 22:977-986 (1994); Eckerand Crooke, BioTechnology 13:351360 (1995), each of which isincorporated herein by reference). The incorporation of non-naturallyoccurring nucleotide analogs or bonds linking the nucleotides or analogscan be particularly useful where the polynucleotide is to be exposed toan environment that can contain a nucleolytic activity, including, forexample, a tissue culture medium or upon administration to a livingsubject, since the modified polynucleotides can be less susceptible todegradation.

A polynucleotide comprising naturally occurring nucleotides andphosphodiester bonds can be chemically synthesized or can be producedusing recombinant DNA methods, using an appropriate polynucleotide as atemplate. In comparison, a polynucleotide comprising nucleotide analogsor covalent bonds other than phosphodiester bonds generally will bechemically synthesized, although an enzyme such as T7 polymerase canincorporate certain types of nucleotide analogs into a polynucleotideand, therefore, can be used to produce such a polynucleotiderecombinantly from an appropriate template (Jellinek et al., supra,1995).

Where a polynucleotide encodes a peptide, for example, a peptide portionof promyostatin or a peptide agent, the coding sequence generally iscontained in a vector and is operatively linked to appropriateregulatory elements, including, if desired, a tissue specific promoteror enhancer. The encoded peptide can be further operatively linked, forexample, to peptide tag such as a His-6 tag or the like, which canfacilitate identification of expression of the agent in the target cell.A polyhistidine tag peptide such as His-6 can be detected using adivalent cation such as nickel ion, cobalt ion, or the like. Additionalpeptide tags include, for example, a FLAG epitope, which can be detectedusing an anti-FLAG antibody (see, for example, Hopp et al.,BioTechnology 6:1204 (1988); U.S. Pat. No. 5,011,912, each of which isincorporated herein by reference); a c-myc epitope, which can bedetected using an antibody specific for the epitope; biotin, which canbe detected using streptavidin or avidin; and glutathione S-transferase,which can be detected using glutathione. Such tags can provide theadditional advantage that they can facilitate isolation of theoperatively linked peptide or peptide agent, for example, where it isdesired to obtain a substantially purified peptide corresponding to aproteolytic fragment of a myostatin polypeptide.

As used herein, the term “operatively linked” or “operativelyassociated” means that two or more molecules are positioned with respectto each other such that they act as a single unit and effect a functionattributable to one or both molecules or a combination thereof. Forexample, a polynucleotide sequence encoding a peptide of the inventioncan be operatively linked to a regulatory element, in which case theregulatory element confers its regulatory effect on the polynucleotidesimilarly to the way in which the regulatory element would effect apolynucleotide sequence with which it normally is associated with in acell. A first polynucleotide coding sequence also can be operativelylinked to a second (or more) coding sequence such that a chimericpolypeptide can be expressed from the operatively linked codingsequences. The chimeric polypeptide can be a fusion polypeptide, inwhich the two (or more) encoded peptides are translated into a singlepolypeptide, i.e., are covalently bound through a peptide bond; or canbe translated as two discrete peptides that, upon translation, canoperatively associate with each other to form a stable complex.

A chimeric polypeptide generally demonstrates some or all of thecharacteristics of each of its peptide components. As such, a chimericpolypeptide can be particularly useful in performing methods of theinvention, as disclosed herein. For example, in one embodiment, a methodof the invention can modulate myostatin signal transduction in a cell.Thus, where one peptide component of a chimeric polypeptide encodes acell compartment localization domain and a second peptide componentencodes a dominant negative Smad polypeptide, the functional chimericpolypeptide can be translocated to the cell compartment designated bythe cell compartment localization domain and can have the dominantnegative activity of the Smad polypeptide, thereby modulating myostatinsignal transduction in the cell.

Cell compartmentalization domains are well known and include, forexample, a plasma membrane localization domain, a nuclear localizationsignal, a mitochondrial membrane localization signal, an endoplasmicreticulum localization signal, or the like (see, for example, Hancock etal., EMBO J. 10:4033-4039, 1991; Buss et al., Mol. Cell. Biol.8:3960-3963, 1988; U.S. Pat. No. 5,776,689 each of which is incorporatedherein by reference). Such a domain can be useful to target an agent toa particular compartment in the cell, or to target the agent forsecretion from a cell. For example, the kinase domain of a myostatinreceptor such as Act RIIB generally is associated with the inner surfaceof the plasma membrane. Thus, a chimeric polypeptide comprising adominant negative myostatin receptor kinase domain, for example, adominant negative Act RIIB receptor, which lacks kinase activity, canfurther comprise a plasma membrane localization domain, therebylocalizing the dominant negative Act RIIB kinase domain to the innercell membrane.

As disclosed herein, a pro-GDF signal peptide has cellular localizationactivity. As used herein, the term “cellular localization activity”refers to the ability of a signal peptide to direct translocation of apeptide operably linked thereto to one or more specific intracellularcompartments or to direct secretion of the molecule from the cell. Assuch, a pro-GDF signal peptide can be particularly useful for directingtranslocation of a peptide or other agent operably linked to the signalpeptide to the same intracellular compartments as a naturally expressedGDF having substantially the same signal peptide. Furthermore, thesignal peptide, for example, a promyostatin signal peptide comprisingabout the first 15 to 30 amino acids of promyostatin, can directsecretion of an operably linked agent from the cell through the samepathway as the naturally occurring pro-GDF having comprising the signalpeptide. Thus, particularly useful agents for performing a method of theinvention include a GDF prodomain or functional peptide portion thereofthat is operably linked to a GDF signal peptide, preferably apromyostatin or pro-GDF-11 signal peptide.

A polynucleotide of the invention, including a polynucleotide agentuseful in performing a method of the invention, can be contacteddirectly with a target cell. For example, oligonucleotides useful asantisense molecules, ribozymes, or triplexing agents can be directlycontacted with a target cell, whereupon the enter the cell and effecttheir function. A polynucleotide agent also can interact specificallywith a polypeptide, for example, a myostatin receptor (or myostatin),thereby altering the ability of myostatin to interact specifically withthe receptor. Such polynucleotides, as well as methods of making andidentifying such polynucleotides, are disclosed herein or otherwise wellknown in the art (see, for example, O□Connell et al., Proc. Natl. Acad.Sci., USA 93:5883-5887, 1996; Tuerk and Gold, Science 249:505-510, 1990;Gold et al., Ann. Rev. Biochem. 64:763-797, 1995; each of which isincorporated herein by reference).

A polynucleotide of the invention, which can encode a peptide portion ofa pro-GDF polypeptide such as promyostatin, or can encode a mutantpromyostatin polypeptide, or can encode a GDF receptor or functionalpeptide portion thereof, or can be a polynucleotide agent useful inperforming a method of the invention, can be contained in a vector,which can facilitate manipulation of the polynucleotide, includingintroduction of the polynucleotide into a target cell. The vector can bea cloning vector, which is useful for maintaining the polynucleotide, orcan be an expression vector, which contains, in addition to thepolynucleotide, regulatory elements useful for expressing thepolynucleotide and, where the polynucleotide encodes a peptide, forexpressing the encoded peptide in a particular cell. An expressionvector can contain the expression elements necessary to achieve, forexample, sustained transcription of the encoding polynucleotide, or theregulatory elements can be operatively linked to the polynucleotideprior to its being cloned into the vector.

An expression vector (or the polynucleotide) generally contains orencodes a promoter sequence, which can provide constitutive or, ifdesired, inducible or tissue specific or developmental stage specificexpression of the encoding polynucleotide, a poly-A recognitionsequence, and a ribosome recognition site or internal ribosome entrysite, or other regulatory elements such as an enhancer, which can betissue specific. The vector also can contain elements required forreplication in a prokaryotic or eukaryotic host system or both, asdesired. Such vectors, which include plasmid vectors and viral vectorssuch as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus,vaccinia virus, semliki forest virus and adeno-associated virus vectors,are well known and can be purchased from a commercial source (Promega,Madison Wis.; Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Md.)or can be constructed by one skilled in the art (see, for example, Meth.Enzymol., Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly,Canc. Gene Ther. 1:51-64, 1994; Flotte, J. Bioenerg. Biomemb. 25:37-42,1993; Kirshenbaum et al., J. Clin. Invest. 92:381-387, 1993; each ofwhich is incorporated herein by reference).

A tetracycline (tet) inducible promoter can be particularly useful fordriving expression of a polynucleotide of the invention, for example, apolynucleotide encoding a dominant negative form of myostatin, in whichthe proteolytic processing site has been mutated, or encoding amyostatin prodomain, which can form a complex with a mature myostatinpeptide, or encoding a dominant negative form of a GDF receptor. Uponadministration of tetracycline, or a tetracycline analog, to a subjectcontaining a polynucleotide operatively linked to a tet induciblepromoter, expression of the encoded peptide is induced, whereby thepeptide can effect its activity, for example, whereby a peptide agentcan reduce or inhibit myostatin signal transduction. Such a method canbe used, for example, to induce muscle hypertrophy in an adult organism.

The polynucleotide also can be operatively linked to tissue specificregulatory element, for example, a muscle cell specific regulatoryelement, such that expression of an encoded peptide is restricted to themuscle cells in an individual, or to muscle cells in a mixed populationof cells in culture, for example, an organ culture. Muscle cell specificregulatory elements including, for example, the muscle creatine kinasepromoter (Sternberg et al., Mol. Cell. Biol. 8:2896-2909, 1988, which isincorporated herein by reference) and the myosin light chainenhancer/promoter (Donoghue et al., Proc. Natl. Acad. Sci., USA88:5847-5851, 1991, which is incorporated herein by reference) are wellknown in the art.

Viral expression vectors can be particularly useful for introducing apolynucleotide into a cell, particularly a cell in a subject. Viralvectors provide the advantage that they can infect host cells withrelatively high efficiency and can infect specific cell types. Forexample, a polynucleotide encoding a myostatin prodomain or functionalpeptide portion thereof can be cloned into a baculovirus vector, whichthen can be used to infect an insect host cell, thereby providing ameans to produce large amounts of the encoded prodomain. The viralvector also can be derived from a virus that infects cells of anorganism of interest, for example, vertebrate host cells such asmammalian, avian or piscine host cells. Viral vectors can beparticularly useful for introducing a polynucleotide useful inperforming a method of the invention into a target cell. Viral vectorshave been developed for use in particular host systems, particularlymammalian systems and include, for example, retroviral vectors, otherlentivirus vectors such as those based on the human immunodeficiencyvirus (HIV), adenovirus vectors, adeno-associated virus vectors,herpesvirus vectors, vaccinia virus vectors, and the like (see Millerand Rosman, BioTechniques 7:980-990, 1992; Anderson et al., Nature392:25-30 Suppl., 1998; Verma and Somia, Nature 389:239-242, 1997;Wilson, New Engl. J. Med. 334:1185-1187 (1996), each of which isincorporated herein by reference).

When retroviruses, for example, are used for gene transfer, replicationcompetent retroviruses theoretically can develop due to recombination ofretroviral vector and viral gene sequences in the packaging cell lineutilized to produce the retroviral vector. Packaging cell lines in whichthe production of replication competent virus by recombination has beenreduced or eliminated can be used to minimize the likelihood that areplication competent retrovirus will be produced. All retroviral vectorsupernatants used to infect cells are screened for replication competentvirus by standard assays such as PCR and reverse transcriptase assays.Retroviral vectors allow for integration of a heterologous gene into ahost cell genome, which allows for the gene to be passed to daughtercells following cell division.

A polynucleotide, which can be contained in a vector, can be introducedinto a cell by any of a variety of methods known in the art (Sambrook etal., Molecular Cloning: A laboratory manual (Cold Spring HarborLaboratory Press 1989); Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1987, and supplementsthrough 1995), each of which is incorporated herein by reference). Suchmethods include, for example, transfection, lipofection, microinjection,electroporation and, with viral vectors, infection; and can include theuse of liposomes, microemulsions or the like, which can facilitateintroduction of the polynucleotide into the cell and can protect thepolynucleotide from degradation prior to its introduction into the cell.The selection of a particular method will depend, for example, on thecell into which the polynucleotide is to be introduced, as well aswhether the cell is isolated in culture, or is in a tissue or organ inculture or in situ.

Introduction of a polynucleotide into a cell by infection with a viralvector is particularly advantageous in that it can efficiently introducethe nucleic acid molecule into a cell ex vivo or in vivo (see, forexample, U.S. Pat. No. 5,399,346, which is incorporated herein byreference). Moreover, viruses are very specialized and can be selectedas vectors based on an ability to infect and propagate in one or a fewspecific cell types. Thus, their natural specificity can be used totarget the nucleic acid molecule contained in the vector to specificcell types. As such, a vector based on an HIV can be used to infect Tcells, a vector based on an adenovirus can be used, for example, toinfect respiratory epithelial cells, a vector based on a herpesvirus canbe used to infect neuronal cells, and the like. Other vectors, such asadeno-associated viruses can have greater host cell range and,therefore, can be used to infect various cell types, although viral ornon-viral vectors also can be modified with specific receptors orligands to alter target specificity through receptor mediated events.

The present invention also provides antibodies that specifically bind apeptide portion of a promyostatin polypeptide or a mutant promyostatinpolypeptide. Particularly useful antibodies of the invention includeantibodies that specifically bind a myostatin prodomain, or a functionalpeptide portion thereof, and antibodies that bind a promyostatinpolypeptide and reduce or inhibit proteolytic cleavage of thepromyostatin to a mature myostatin peptide. In addition, an antibody ofthe invention can be an antibody that specifically binds a GDF receptor,or functional peptide portion thereof, as described below. Methods ofpreparing and isolating an antibody of the invention are described ingreater detail below, the disclosure of which is incorporated herein byreference.

Myostatin is essential for proper regulation of skeletal muscle mass. Ascompared to wild type mice, myostatin knock-out mice, which lackmyostatin, have two to three times the amount of muscle due to acombination of hyperplasia and hypertrophy. As disclosed herein,myostatin knock-out mice also have a dramatic reduction in fataccumulation due, at least in part, to an increased anabolic state ofskeletal muscle tissue throughout the body. Conversely, overexpressionof myostatin in nude mice induced a wasting syndrome that resembles thecachectic state observed in human patients suffering from chronicdiseases such as cancer or AIDS. As further disclosed herein, myostatinactivity can be mediated through a signal transduction having thecharacteristics of the Smad signal transduction pathway. Accordingly,the invention provides methods of modulating an effect of myostatin on acell by contacting the cell with an agent that affects myostatin signaltransduction in the cell.

As used herein, the term “modulate,” when used in reference to an effectof myostatin on a cell, means that myostatin signal transduction in thecell either is increased or is reduced or inhibited. The terms“increase” and “reduce or inhibit” are used in reference to a baselinelevel of myostatin signal transduction activity, which can be the levelof activity of the signal transduction pathway in the absence ofmyostatin, or the level of activity in a normal cell in the presence ofmyostatin. For example, the myostatin signal transduction pathwayexhibits a particular activity in a muscle cell contacted withmyostatin, and, upon further contacting the muscle cell with a myostatinprodomain, myostatin signal transduction activity can be reduced orinhibited. As such, a myostatin prodomain is an agent useful forreducing or inhibiting myostatin signal transduction. Similarly, aprodomain of another GDF family member such as a GDF-11 prodomain, or ofanother TGF-θ family member such as an activin prodomain, MIS prodomain,or the like, can be useful for reducing myostatin signal transduction.The terms “reduce or inhibit” are used together herein because it isrecognized that, in some cases, the level of myostatin signaltransduction can be reduced below a level that can be detected by aparticular assay. As such, it may not be determinable using such anassay as to whether a low level of myostatin signal transductionremains, or whether the signal transduction is completely inhibited.

As used herein, the term “myostatin signal transduction” refers to theseries of events, generally a series of protein-protein interactions,that occurs in a cell due to the specific interaction of myostatin witha myostatin receptor expressed on the surface of the cell. As such,myostatin signal transduction can be detected, for example, by detectinga specific interaction of myostatin with its receptor on a cell, bydetecting phosphorylation of one or more polypeptides involved in amyostatin signal transduction pathway in the cell, by detectingexpression of one or more genes that are specifically induced due tomyostatin signal transduction, or by detecting a phenotypic change thatoccurs in response to myostatin signal transduction (see Examples). Asdisclosed herein, an agent useful in a method of the invention can actas an agonist to stimulate myostatin signal transduction or as anantagonist to reduce or inhibit myostatin signal transduction.

The methods of the present invention are exemplified generally hereinwith respect to myostatin. It should be recognized, however, that themethods of the invention can more broadly encompass modulating an effectof other GDF peptides, for example, GDF-11, on a cell by contacting thecell with an agent that affects signal transduction due to the GDF inthe cell. Methods of practicing the full scope of the invention will bereadily known in view of the present disclosure, which includes, forexample, methods for identifying GDF receptors, methods for identifyingagents that modulate signal transduction due to a specific interactionof the GDF with its receptor, and the like.

A myostatin signal transduction pathway is exemplified herein by theSmad pathway, which is initiated upon myostatin specifically interactingwith the extracellular domain of an activin type II receptor andpropagated through interactions of intracellular polypeptides, includingSmad proteins, in the cell. In general, myostatin signal transduction isassociated with phosphorylation or dephosphorylation of specificintracellular polypeptides such as Smad polypeptides. Thus, myostatinsignal transduction in a cell can be detected by detecting an increasedlevel of phosphorylation of one or more Smad polypeptides in thepresence of myostatin as compared to the level of phosphorylation of thepolypeptides in the absence of myostatin. A method of the inventionprovides a means to increase or decrease myostatin signal transductionand, therefore, the level of phosphorylation of an Smad polypeptideinvolved in a myostatin signal transduction pathway will be increasedabove a normal level or decreased below a level expected in the presenceof myostatin, respectively.

A method of the invention can be performed, for example, by contactingunder suitable conditions a target cell and an agent that affectsmyostatin signal transduction in the cell. Suitable conditions can beprovided by placing the cell, which can be an isolated cell or can be acomponent of a tissue or organ, in an appropriate culture medium, or bycontacting the cell in situ in an organism. For example, a mediumcontaining the cell can be contacted with an agent the affects theability of myostatin to specifically interact with a myostatin receptorexpressed on the cell, or with an agent that affects a myostatin signaltransduction pathway in the cell. In general, the cell is a component ofa tissue or organ in a subject, in which case contacting the cell cancomprise administering the agent to the subject. However, the cell alsocan be manipulated in culture, then can be maintained in culture,administered to a subject, or used to produce a transgenic nonhumananimal.

An agent useful in a method of the invention can be any type ofmolecule, for example, a polynucleotide, a peptide, a peptidomimetic,peptoids such as vinylogous peptoids, a small organic molecule, or thelike, and can act in any of various ways to affect myostatin signaltransduction. The agent can act extracellularly by binding myostatin ora myostatin receptor such as an activin receptor, thereby altering theability of myostatin to specifically interact with its receptor, or canact intracellularly to alter myostatin signal transduction in the cell.In addition, the agent can be an agonist, which mimics or enhances theeffect of myostatin on a cell, for example, the ability of myostatin tospecifically interact with its receptor, thereby increasing myostatinsignal transduction in the cell; or can be an antagonist, which canreduces or inhibits the effect of myostatin on a cell, thereby reducingor inhibiting myostatin signal transduction in the cell.

As used herein, the term “specific interaction” or “specifically binds”or the like means that two molecules form a complex that is relativelystable under physiologic conditions. The term is used herein inreference to various interactions, including, for example, theinteraction of myostatin and a myostatin receptor, the interaction ofthe intracellular components of a myostatin signal transduction pathway,the interaction of an antibody and its antigen, and the interaction of amyostatin prodomain with myostatin. A specific interaction can becharacterized by a dissociation constant of at least about 1×10⁻⁶ M,generally at least about 1×10-7 M, usually at least about 1×10-8 M, andparticularly at least about 1×10-9 M or 1×10-10 M or greater. A specificinteraction generally is stable under physiological conditions,including, for example, conditions that occur in a living individualsuch as a human or other vertebrate or invertebrate, as well asconditions that occur in a cell culture such as used for maintainingmammalian cells or cells from another vertebrate organism or aninvertebrate organism. In addition, a specific interaction such as theextracellular interaction of a myostatin prodomain and myostatingenerally is stable under conditions such as those used for aquacultureof a commercially valuable marine organism. Methods for determiningwhether two molecules interact specifically are well known and include,for example, equilibrium dialysis, surface plasmon resonance, and thelike.

An agent that alters a specific interaction of myostatin with itsreceptor can act, for example, by binding to myostatin such that itcannot interact specifically with its cellular receptor, by competingwith myostatin for binding to its receptor, or by otherwise by-passingthe requirement that myostatin specifically interact with its receptorin order to induce myostatin signal transduction. A truncated myostatinreceptor such as a soluble extracellular domain of a myostatin receptoris an example of an agent that can bind myostatin, thereby sequesteringmyostatin and reducing or inhibiting its ability to interactspecifically with a cell surface myostatin receptor. A myostatinprodomain or functional peptide portion thereof is another example of anagent that can bind myostatin, thereby reducing or inhibiting theability of the myostatin to interact specifically with a cell surfacemyostatin receptor. Such myostatin antagonists are useful in practicinga method of the invention, particularly for reducing or inhibitingmyostatin signal transduction in a cell.

Follistatin is another example of an agent that can bind to myostatin,thereby reducing or inhibiting the ability of myostatin to interactspecifically with its receptor. Follistatin can bind to and inhibit theactivity of various TGF-β family members, including myostatin (GDF-8;U.S. Pat. No. 6,004,937) and GDF-11 (Gamer et al., Devel. Biol.208:222-232, 1999) and, therefore, can be used for performing a methodas disclosed. Although the use of follistatin for modulating the effectsof myostatin previously has been described (U.S. Pat. No. 6,004,937), itwas not known, prior to the present disclosure, that follistatin reducesor inhibits the ability of myostatin to interact specifically with amyostatin receptor such as Act RIIB.

An agent useful in a method of the invention also can interact with acellular myostatin receptor, thereby competing with myostatin for thereceptor. Such an agent can be, for example, an antibody thatspecifically binds a cell surface myostatin receptor, including all or aportion of the myostatin binding domain, thereby preventing myostatinfrom interacting specifically with the receptor. Such an anti-myostatinreceptor antibody can be selected for its ability to specifically bindthe receptor without activating myostatin signal transduction and,therefore, can be useful as a myostatin antagonist for reducing orinhibiting myostatin signal transduction; or can be selected for itsability to specifically bind the receptor and activate myostatin signaltransduction, thus acting as a myostatin agonist. The antibody can beraised using a myostatin receptor, or the extracellular domain of thereceptor, as an immunogen, or can be an anti-idiotype antibody, which israised against an anti-myostatin antibody and mimics myostatin. Anti-GDFreceptor antibodies are discussed in greater detail below.

An agent useful in a method of the invention also can be an agent thatreduces or inhibits proteolytic cleavage of a pro-GDF polypeptide to anactive mature GDF peptide, thereby reducing or inhibiting GDF signaltransduction. Such an agent can be a protease inhibitor, particularlyone that inhibits the activity of a protease that recognizes and cleavesan Arg-Xaa-Xaa-Arg (SEQ ID NO: 21) proteolytic recognition site. Wherethe pro-GDF is promyostatin, an anti-myostatin antibody that reduces orinhibits the specific binding of a protease to the Arg-Xaa-Xaa-Arg (SEQID NO: 21) proteolytic cleavage site in myostatin also can be usedreduce or inhibit proteolysis of promyostatin, thereby reducing theamount of mature myostatin produced. Such an antibody can bind theproteolytic cleavage site, or can bind some other site on the pro-GDFpolypeptide such that binding of and cleavage by the protease is reducedor inhibited.

In addition, an agent useful in a method of the invention can be amutant myostatin receptor, which, for example, lacks myostatin signaltransduction activity in response to myostatin binding, or hasconstitutive myostatin signal transduction activity. For example, amutant myostatin receptor can have a point mutation, a deletion, or thelike in its kinase domain such that the receptor lacks kinase activity.Such a dominant negative mutant myostatin receptor lacks the ability totransmit myostatin signal transduction despite the fact that it canspecifically bind myostatin.

An agent useful in a method of the invention also can modulate the levelor activity of an intracellular polypeptide involved in a myostatinsignal transduction pathway. As disclosed herein, regulation of musclegrowth by myostatin can involve components of a signal transductionpathway that is activated by activin type II receptors (see Examples 7and 9; see, also, Example 14). Myostatin specifically interacts withactivin type IIB receptors (Act RIIB) expressed in COS cells in culture(Example 7). The low affinity of binding indicates that myostatinbinding to Act RIIB in vivo may involve other factors, similar to TGF-β,which has significantly higher affinity for the type II receptor whenthe type I receptor also is present (Attisano et al., Cell 75:671-680,1993), or to other systems that require other molecules for presentingthe ligand to the signaling receptor (Massague, supra, 1998; Wang etal., Cell 67:795-805, 1991).

The specific interaction of myostatin with Act RIIB indicates thatmyostatin signal transduction can involve components of the Smad signaltransduction pathway. Thus, the Smad signal transduction pathwayprovides a target for modulating the effect of myostatin on a cell, andagents that affect the Smad pathway can be useful for modulatingmyostatin signal transduction in a cell.

Agents useful for modulating the level or activity of intracellularpolypeptide components of a GDF signal transduction include agonists,which can increase signal transduction activity, and antagonists, whichcan reduce or inhibit signal transduction activity. With respect tomyostatin, for example, agents that can increase myostatin signaltransduction activity are exemplified by phosphatase inhibitors, whichcan reduce or inhibit dephosphorylation of Smad polypeptides, therebyprolonging the signal transducing activity of the Smad. Dominantnegative Smad 6 or Smad 7 polypeptides, which can negate the inhibitoryeffect of Smad 6 and Smad 7 on myostatin signal transduction, areadditional examples of agents that can increase myostatin signaltransduction activity by increasing Smad signal transduction.

Antagonist agents that can reduce or inhibit myostatin signaltransduction activity are exemplified by dominant negative Smadpolypeptides such as dominant negative Smad 2, Smad 3 or Smad 4, inwhich the C-terminal phosphorylation sites have been mutated. Theinhibitory Smad polypeptides such as Smad 6 and Smad 7, which inhibitthe activation of Smad 2 and Smad 3; and a c-ski polypeptide, whichbinds Smad polypeptides and inhibits signal transduction, are additionalexamples of antagonists useful for reducing or inhibiting myostatinsignal transduction by decreasing Smad signal transduction.

Where the agent that acts intracellularly is a peptide, it can becontacted with the cell directly, or a polynucleotide encoding thepeptide (or polypeptide) can be introduced into the cell and the peptidecan be expressed in the cell. It is recognized that some of the peptidesuseful in a method of the invention are relatively large and, therefore,may not readily traverse a cell membrane. However, various methods areknown for introducing a peptide into a cell. The selection of a methodfor introducing such a peptide into a cell will depend, in part, on thecharacteristics of the target cell, into which the polypeptide is to beprovided. For example, where the target cells, or a few cell typesincluding the target cells, express a receptor, which, upon binding aparticular ligand, is internalized into the cell, the peptide agent canbe operatively associated with the ligand. Upon binding to the receptor,the peptide is translocated into the cell by receptor-mediatedendocytosis. The peptide agent also can be encapsulated in a liposome orformulated in a lipid complex, which can facilitate entry of the peptideinto the cell, and can be further modified to express a receptor (orligand), as above. The peptide agent also can be introduced into a cellby engineering the peptide to contain a protein transduction domain suchas the human immunodeficiency virus TAT protein transduction domain,which facilitates translocation of the peptide into the cell (seeSchwarze et al., Science 285:1569-1572 (1999), which is incorporatedherein by reference; see, also, Derossi et al., J. Biol. Chem. 271:18188(1996)).

The target cell also can be contacted with a polynucleotide encoding thepeptide agent, which can be expressed in the cell. The expressed peptideagent can be a mutant GDF receptor or peptide portion thereof. Exampleof a mutant GDF receptor include a kinase-deficient form of a myostatinreceptor such as a dominant negative Act RIIA or Act RIIB, which can,but need not, have the ability to specifically bind a ligand (e.g.,myostatin); and a truncated myostatin or other GDF receptor, such as asoluble form of a myostatin receptor, which binds myostatin, therebysequestering it from interacting specifically with a cellular myostatinreceptor; a dominant-negative form of a Smad polypeptide such as adominant negative Smad 3, in which the C-terminal phosphorylation siteshave been mutated (Liu et al., Proc. Natl. Acad. Sci., USA94:10669-10674, 1997); a Smad 7 polypeptide, which inhibits theactivation of Smad 2 and Smad 3 (Heldin et al., Nature 390:465-471,1997); or a c-ski polypeptide, which can bind a Smad polypeptide andinhibit signal transduction by the Smad (Suprave et al., Genes Devel.4:1462-1472, 1990).

Expression of a c-ski peptide agent in a cell can be particularly usefulfor modulating myostatin signal transduction. Mice lacking c-ski show asevere reduction in skeletal muscle mass (Berk et al., Genes Devel.11:2029-2039, 1997), whereas transgenic mice that overexpress c-ski inmuscle show dramatic muscle hypertrophy (Suprave et al., supra, 1990).c-ski interacts with and blocks the activity of certain Smad proteins,including Smad 2, Smad 3 and Smad 4, which mediate signaling of TGF-βand activin type II receptors (Luo et al., Genes Devel. 13:2196-1106,1999; Stroschein et al., Science 286:771-774, 1999; Sun et al., Mol.Cell. 4:499-509, 1999a; Sun et al., Proc. Natl. Acad. Sci., USA96:112442-12447, 1999b; Akiyoshi et al., J. Biol. Chem. 274:35269,1999). Thus, in view of the present disclosure that myostatin activitycan be mediated through Act RIIB binding, it will be recognized that theactivity of myostatin, or of any GDF that utilizes an Smad pathway, canbe modulated by increasing or decreasing c-ski expression in a targetcell.

An agent useful in a method of the invention can be a polynucleotide,which can be contacted with or introduced into a cell as describedabove. Generally, but not necessarily, the polynucleotide is introducedinto the cell, where it effects its function either directly, orfollowing transcription or translation or both. For example, asdiscussed above, the polynucleotide can encode a peptide agent, which isexpressed in the cell and modulates myostatin activity. Such anexpressed peptide can be, for example, a mutant promyostatinpolypeptide, which cannot be cleaved into active myostatin; or can be amutant myostatin receptor, for example, a truncated myostatin receptorextracellular domain; a myostatin receptor extracellular domainoperatively associated with a membrane anchoring domain; or a mutantmyostatin receptor lacking protein kinase activity. Methods forintroducing a polynucleotide into a cell are exemplified below orotherwise known in the art.

A polynucleotide agent useful in a method of the invention also can be,or can encode, an antisense molecule, a ribozyme or a triplexing agent.For example, the polynucleotide can be (or can encode) an antisensenucleotide sequence such as an antisense c-ski nucleotide sequence,which can act as an agonist to increase myostatin signal transduction ina cell; or an antisense Smad nucleotide sequence, which can act eitheras an agonist to increase myostatin signal transduction or as antagonistto reduce or inhibit myostatin signal transduction, depending on theparticular Smad antisense nucleotide sequence. Such polynucleotides canbe contacted directly with a target cell and, upon uptake by the cell,can effect their antisense, ribozyme or triplexing activity; or can beencoded by a polynucleotide that is introduced into a cell, whereuponthe polynucleotide is expressed to produce, for example, an antisenseRNA molecule or ribozyme, which effects its activity.

An antisense polynucleotide, ribozyme or triplexing agent iscomplementary to a target sequence, which can be a DNA or RNA sequence,for example, messenger RNA, and can be a coding sequence, a nucleotidesequence comprising an intron-exon junction, a regulatory sequence suchas a Shine-Delgarno sequence, or the like. The degree of complementarityis such that the polynucleotide, for example, an antisensepolynucleotide, can interact specifically with the target sequence in acell. Depending on the total length of the antisense or otherpolynucleotide, one or a few mismatches with respect to the targetsequence can be tolerated without losing the specificity of thepolynucleotide for its target sequence. Thus, few if any mismatcheswould be tolerated in an antisense molecule consisting, for example, of20 nucleotides, whereas several mismatches will not affect thehybridization efficiency of an antisense molecule that is complementary,for example, to the full length of a target mRNA encoding a cellularpolypeptide. The number of mismatches that can be tolerated can beestimated, for example, using well known formulas for determininghybridization kinetics (see Sambrook et al., supra, 1989) or can bedetermined empirically using methods as disclosed herein or otherwiseknown in the art, particularly by determining that the presence of theantisense polynucleotide, ribozyme, or triplexing agent in a celldecreases the level of the target sequence or the expression of apolypeptide encoded by the target sequence in the cell.

A polynucleotide useful as an antisense molecule, a ribozyme or atriplexing agent can inhibit translation or cleave the nucleic acidmolecule, thereby modulating myostatin signal transduction in a cell. Anantisense molecule, for example, can bind to an mRNA to form a doublestranded molecule that cannot be translated in a cell. Antisenseoligonucleotides of at least about 15 to 25 nucleotides are preferredsince they are easily synthesized and can hybridize specifically with atarget sequence, although longer antisense molecules can be expressedfrom a polynucleotide introduced into the target cell. Specificnucleotide sequences useful as antisense molecules can be identifiedusing well known methods, for example, gene walking methods (see, forexample, Seimiya et al., J. Biol. Chem. 272:4631-4636 (1997), which isincorporated herein by reference). Where the antisense molecule iscontacted directly with a target cell, it can be operatively associatedwith a chemically reactive group such as iron-linked EDTA, which cleavesa target RNA at the site of hybridization. A triplexing agent, incomparison, can stall transcription (Maher et al., Antisense Res. Devel.1:227 (1991); Helene, Anticancer Drug Design 6:569 (1991)). Thus, atriplexing agent can be designed to recognize, for example, a sequenceof a Smad gene regulatory element, thereby reducing or inhibiting theexpression of a Smad polypeptide in the cell, thereby modulatingmyostatin signal transduction in a target cell.

The present invention also provides a method of identifying an agentthat can alter the effect of a GDF such myostatin on a cell,particularly agents that can alter the ability of the GDF to interactspecifically with its cellular receptor. Such agents can act byincreasing or decreasing the ability of the GDF to interact specificallywith its receptor and, therefore, are useful for increasing ordecreasing GDF signal transduction, respectively. A screening method ofthe invention is exemplified herein using a myostatin receptor, forexample, an activin type II receptor such as Act RIIA or Act RIIB.

A screening method of the invention can be performed, for example, bycontacting under suitable conditions myostatin, or a functional peptideportion thereof, a myostatin receptor such as Act RIIA or Act RIIB, andan agent to be tested. The myostatin, the receptor and the agent can becontacted in any order as desired. As such, the screening method can beused to identify agents that can competitively or non-competitivelyinhibit myostatin binding to the receptor, agents that can mediate orenhance myostatin binding to the receptor, agents that can inducedissociation of specifically bound myostatin from the receptor, andagents that otherwise affect the ability of myostatin to induce signaltransduction, such agents having agonist or antagonist activity.Appropriate control reactions are performed to confirm that the actionof the agent is specific with respect to the myostatin or other GDFreceptor.

Suitable conditions for performing a screening method of the inventioncan be any conditions that allow myostatin to specifically interact withits receptor, including methods as disclosed herein (see Examples 7 and9) or otherwise known in the art. Thus, suitable conditions forperforming the screening assay can be, for example, in vitro conditionsusing a substantially purified myostatin receptor; cell cultureconditions, utilizing a cell that normally expresses a myostatinreceptor, for example, an adipocyte or a muscle cell, or a cell that hasbeen genetically modified to express a functional myostatin receptor onits surface; or in situ conditions as occur in an organism.

A screening method of the invention also can be performed using themethods of molecular modeling as described above. The utilization of amolecular modeling method provides a convenient, cost effective means toidentify those agents, among a large population such as a combinatoriallibrary of potential agents, that are most likely to interactspecifically with a GDF receptor, thereby reducing the number ofpotential agents that need to be screened using a biological assay. Uponidentifying agents that interact specifically with a GDF receptor suchas Act RIIB using a molecular modeling method, the selected agents canbe examined for the ability to modulate an effect of a GDF such asmyostatin on a cell using the methods disclosed herein.

The ability of a test agent to modulate an effect of myostatin can bedetected using methods as disclosed herein (see Examples 7 and 9) orotherwise known in the art. The term “test agent” or “test molecule” isused broadly herein to mean any agent that is being examined for agonistor antagonist activity in a method of the invention. Although the methodgenerally is used as a screening assay to identify previously unknownmolecules that can act as agonist or antagonist agents as describedherein, the methods also can be used to confirm that a agent known tohave a particular activity in fact has the activity, for example, instandardizing the activity of the agent.

A method of the invention can be performed, for example, by contactingmyostatin with a cell that has been genetically modified to express anAct RIIB receptor, and determining the effect of an agent, for example,a dominant negative Act RIIB, by examining phosphorylation of a Smadpolypeptide involved in the myostatin signal transduction pathway. Ifdesired, the cell can be further genetically modified to contain areporter nucleotide sequence, the expression of which is dependent onthe myostatin signal transduction pathway, for example, on activation ofthe Smad pathway, and the effect of the test agent can be determined bycomparing expression of the reporter nucleotide sequence in the presenceand absence of the agent, the myostatin, or both. Expression of thereporter nucleotide sequence can be detected, for example, by detectingan RNA transcript of the reporter nucleotide sequence, or by detecting apolypeptide encoded by the reporter nucleotide sequence. A polypeptidereporter can be, for example, a θ-lactamase, chloramphenicolacetyltransferase, adenosine deaminase, aminoglycosidephosphotransferase, dihydrofolate reductase, hygromycin-Bphosphotransferase, thymidine kinase, θ-galactosidase, luciferase orxanthine guanine phosphoribosyltransferase polypeptide or the like, andcan be detected, for example, by detecting radioactivity, luminescence,chemiluminescence, fluorescence, enzymatic activity, or specific bindingdue to the reporter polypeptide.

A screening method of the invention provides the advantage that it canbe adapted to high throughput analysis and, therefore, can be used toscreen combinatorial libraries of test agents in order to identify thoseagents that can modulate an effect of myostatin on a cell, includingthose agents that can alter a specific interaction of myostatin and amyostatin receptor. Methods for preparing a combinatorial library ofmolecules that can be tested for a desired activity are well known inthe art and include, for example, methods of making a phage displaylibrary of peptides, which can be constrained peptides (see, forexample, U.S. Pat. No. 5,622,699; U.S. Pat. No. 5,206,347; Scott andSmith, Science 249:386-390, 1992; Markland et al., Gene 109:13-19, 1991;each of which is incorporated herein by reference); a peptide library(U.S. Pat. No. 5,264,563, which is incorporated herein by reference); apeptidomimetic library (Blondelle et al., Trends Anal. Chem. 14:83-92,1995; a nucleic acid library (O'Connell et al., supra, 1996; Tuerk andGold, supra, 1990; Gold et al., supra, 1995; each of which isincorporated herein by reference); an oligosaccharide library (York etal., Carb. Res., 285:99-128, 1996; Liang et al., Science, 274:1520-1522,1996; Ding et al., Adv. Expt. Med. Biol., 376:261-269, 1995; each ofwhich is incorporated herein by reference); a lipoprotein library (deKruif et al., FEBS Lett., 399:232-236, 1996, which is incorporatedherein by reference); a glycoprotein or glycolipid library (Karaoglu etal., J. Cell Biol., 130:567-577, 1995, which is incorporated herein byreference); or a chemical library containing, for example, drugs orother pharmaceutical agents (Gordon et al., J. Med. Chem., 37:1385-1401,1994; Ecker and Crooke, Bio/Technology, 13:351-360, 1995; each of whichis incorporated herein by reference). Polynucleotides can beparticularly useful as agents that can modulate a specific interactionof myostatin and its receptor because nucleic acid molecules havingbinding specificity for cellular targets, including cellularpolypeptides, exist naturally, and because synthetic molecules havingsuch specificity can be readily prepared and identified (see, forexample, U.S. Pat. No. 5,750,342, which is incorporated herein byreference).

In view of the present disclosure, it will be recognized that variousanimal model systems can be used as research tools to identify agentsuseful for practicing a method of the invention. For example, transgenicmice or other experimental animals can be prepared using the variousmyostatin inhibitor constructs disclosed herein, and the transgenicnon-human organism can be examined directly to determine the effectproduced by expressing various levels of a particular agent in theorganism. In addition, the transgenic organism, for example, atransgenic mouse, can be crossbred with other mice, for example, withob/ob, db/db, or agouti lethal yellow mutant mice, to determine optimallevels of expression of a myostatin inhibitor useful for treating orpreventing a disorder such as obesity, type II diabetes, or the like. Assuch, the present invention provides transgenic non-human organisms,particularly transgenic organisms containing a polynucleotide encoding amyostatin prodomain, which can include the myostatin signal peptide, apro-peptide (see Examples) or a polynucleotide encoding a mutantpromyostatin polypeptide. Further, the invention provides transgenicnon-human organisms that express high levels of follistatin or thatexpress a dominant negative Act IIB receptor (see Examples). Suchorganisms exhibit dramatic increases in muscle mass, similar to thatseen in myostatin knock-out mice (see for example, U.S. Pat. No.5,994,618, herein incorporated by reference). As discussed herein, suchanimal models are important to identify agents for enhancing musclegrowth for therapeutic purposes and agricultural applications. Methodsof producing transgenic non-human animals are known in the art (see forExample U.S. Pat. Nos. 6,140,552; 5,998,698; 6,218,596, all of which areherein incorporated by reference).

A myostatin polynucleotide of the invention is derived from anyorganism, including mouse, rat, cow, pig, human, chicken, ovine, turkey,finfish and other aquatic organisms and other species. Suchpolynucleotides can be used to make transgenic animals as describedherein. Examples of aquatic animals for which transgenics can be made(and from which myostatin polynucleotide can be derived) include thosebelonging to the class Piscine such as salmon, trout, char, ayu, carp,crucian carp, goldfish, roach, whitebait, eel, conger eel, sardine,zebrafish, flying fish, sea bass, sea bream, parrot bass, snapper,mackerel, horse mackerel, tuna, bonito, yellowtail, rockfish, fluke,sole, flounder, blowfish, filefish; those belonging to the classCephalopoda such as squid, cuttlefish, octopus; those belonging to theclass Pelecypoda such as clam (e.g., hardshell, Manila, Quahog, Surf,Soft-shell); cockles, mussels, periwinkles; scallops (e.g., sea, bay,calloo); conch, snails, sea cucumbers; ark shell; oyster (e.g., C.virginica, Gulf, New Zealand, Pacific); those belonging to the classGastropoda such as turban shell, abalone (e.g. green, pink, red); andthose belonging to the class Crustacea such as lobster, including butnot limited to Spiny, Rock, and American; prawn; shrimp, including butnot limited to M. rosenbergii, P. styllrolls, P. indicus, P. jeponious,P. monodon, P. vannemel, M. ensis, S. melantho, N. norvegious, coldwater shrimp; crab, including but not limited to Blue, rook, stone,king, queen, snow, brown, dungeness, Jonah, Mangrove, soft-shelled,squilla, krill, langostinos; crayfish/crawfish, including but notlimited to Blue, Marron, Red Claw, Red Swamp, Soft-shelled, white;Annelida; Chordates, including but not limited to reptiles, such asalligators and turtles; and Amphibians, including frogs; andEchinoderms, including but not limited to sea urchins.

Various methods are known for producing a transgenic animal. In onemethod, an embryo at the pronuclear stage (a “one cell embryo”) isharvested from a female and the transgene is microinjected into theembryo, in which case the transgene will be chromosomally integratedinto the germ cells and somatic cells of the resulting mature animal. Inanother method, embryonic stem cells are isolated and the transgene isincorporated into the stem cells by electroporation, plasmidtransfection or microinjection; the stem cells are then reintroducedinto the embryo, where they colonize and contribute to the germ line.Methods for microinjection of polynucleotides into mammalian species aredescribed, for example, in U.S. Pat. No. 4,873,191, which isincorporated herein by reference. In yet another method, embryonic cellsare infected with a retrovirus containing the transgene, whereby thegerm cells of the embryo have the transgene chromosomally integratedtherein.

When the animals to be made transgenic are avian, microinjection intothe pronucleus of the fertilized egg is problematic because avianfertilized ova generally go through cell division for the first twentyhours in the oviduct and, therefore, the pronucleus is inaccessible.Thus, the retrovirus infection method is preferred for making transgenicavian species (see U.S. Pat. No. 5,162,215, which is incorporated hereinby reference). If microinjection is to be used with avian species,however, the embryo can be obtained from a sacrificed hen approximately2.5 hours after the laying of the previous laid egg, the transgene ismicroinjected into the cytoplasm of the germinal disc and the embryo iscultured in a host shell until maturity (Love et al., Biotechnology 12,1994). When the animals to be made transgenic are bovine or porcine,microinjection can be hampered by the opacity of the ova, thereby makingthe nuclei difficult to identify by traditional differentialinterference-contrast microscopy. To overcome this problem, the ovafirst can be centrifuged to segregate the pronuclei for bettervisualization.

Non-human transgenic animals of the invention can be bovine, porcine,ovine, piscine, avian or other animals. The transgene can be introducedinto embryonal target cells at various developmental stages, anddifferent methods are selected depending on the stage of development ofthe embryonal target cell. The zygote is the best target formicroinjection. The use of zygotes as a target for gene transfer has amajor advantage in that the injected DNA can incorporate into the hostgene before the first cleavage (Brinster et al., Proc. Natl. Acad. Sci.,USA 82:4438-4442, 1985). As a consequence, all cells of the transgenicnon-human animal carry the incorporated transgene, thus contributing toefficient transmission of the transgene to offspring of the founder,since 50% of the germ cells will harbor the transgene.

A transgenic animal can be produced by crossbreeding two chimericanimals, each of which includes exogenous genetic material within cellsused in reproduction. Twenty-five percent of the resulting offspringwill be transgenic animals that are homozygous for the exogenous geneticmaterial, 50% of the resulting animals will be heterozygous, and theremaining 25% will lack the exogenous genetic material and have a wildtype phenotype.

In the microinjection method, the transgene is digested and purifiedfree from any vector DNA, for example, by gel electrophoresis. Thetransgene can include an operatively associated promoter, whichinteracts with cellular proteins involved in transcription, and providesfor constitutive expression, tissue specific expression, developmentalstage specific expression, or the like. Such promoters include thosefrom cytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpesvirus, as well as those from the genes encoding metallothionein,skeletal actin, Phosphenolpyruvate carboxylase (PEPCK), phosphoglycerate(PGK), dihydrofolate reductase (DHFR), and thymidine kinase (TK).Promoters from viral long terminal repeats (LTRs) such as Rous sarcomavirus LTR also can be employed. When the animals to be made transgenicare avian, preferred promoters include those for the chicken □-globingene, chicken lysozyme gene, and avian leukosis virus. Constructs usefulin plasmid transfection of embryonic stem cells will employ additionalregulatory elements, including, for example, enhancer elements tostimulate transcription, splice acceptors, termination andpolyadenylation signals, ribosome binding sites to permit translation,and the like.

In the retroviral infection method, the developing non-human embryo canbe cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retroviral infection (Jaenich, Proc.Natl. Acad. Sci, USA 73:1260-1264, 1976). Efficient infection of theblastomeres is obtained by enzymatic treatment to remove the zonapellucida (Hogan et al., Manipulating the Mouse Embryo (Cold SpringHarbor Laboratory Press, 1986). The viral vector system used tointroduce the transgene is typically a replication-defective retroviruscarrying the transgene (Jahner et al., Proc. Natl. Acad. Sci., USA82:6927-6931, 1985; Van der Putten et al., Proc. Natl. Acad. Sci, USA82:6148-6152, 1985). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus producing cells (Vander Putten et al., supra, 1985; Stewart et al., EMBO J. 6:383-388,1987). Alternatively, infection can be performed at a later stage. Virusor virus-producing cells can be injected into the blastocoele (Jahner etal., Nature 298:623-628, 1982). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic nonhuman animal. Further, the founder cancontain various retroviral insertions of the transgene at differentpositions in the genome, which generally will segregate in theoffspring. In addition, it is also possible to introduce transgenes intothe germ line, albeit with low efficiency, by intrauterine retroviralinfection of the mid-gestation embryo (Jahner et al., supra, 1982).

Embryonal stem cell (ES) also can be targeted for introduction of thetransgene. ES cells are obtained from pre-implantation embryos culturedin vitro and fused with embryos (Evans et al. Nature 292:154-156, 1981;Bradley et al., Nature 309:255-258, 1984; Gossler et al., Proc. Natl.Acad. Sci., USA 83:9065-9069, 1986; Robertson et al., Nature322:445-448, 1986). Transgenes can be efficiently introduced into the EScells by DNA transfection or by retrovirus mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anonhuman animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal (seeJaenisch, Science 240:1468-1474, 1988).

As disclosed herein, myostatin can exert its activity, at least in part,through the Smad signal transduction pathway, and myostatin expressioncan be associated with various pathological conditions. As such, thepresent invention provides new targets for the treatment of variouspathological conditions associated with myostatin, including metabolicconditions such as obesity and type II diabetes. Accordingly, thepresent invention provides methods for ameliorating the severity of apathological condition in a subject, wherein the pathologic condition ischaracterized at least in part by an abnormal amount, development ormetabolic activity of muscle or adipose tissue, by modulating myostatinsignal transduction in a muscle cell or adipose tissue cell in thesubject.

Myostatin functions as a negative regulator of muscle growth (McPherronet al., supra, 1997). Myostatin knock-out mice weighed approximately 25%to 30% more than wild type littermates, and this increase in body weightin the mice examined resulted entirely from a dramatic increase inskeletal muscle tissue weight. In mice lacking myostatin, the skeletalmuscles weighed about 2 to 3 times as much as the corresponding musclesof wild type littermates. This increased muscle weight in the homozygousknock-out mice resulted from a combination of hyperplasia andhypertrophy.

As disclosed herein, heterozygous myostatin knock-out mice also haveincreased skeletal muscle mass, although to a lesser extent than thatobserved in homozygous mutant mice, thus demonstrating that myostatinacts in a dose-dependent manner in vivo (see Example 1). Furthermore,overexpression of myostatin in animals had the opposite effect withrespect to muscle growth. For example, nude mice carryingmyostatin-expressing tumors developed a wasting syndrome characterizedby a dramatic loss of muscle and fat weight (see Example 8). Thissyndrome in the nude mice resembled the cachectic state that occurs inpatients with chronic diseases such as cancer or AIDS.

The serum levels of myostatin immunoreactive material have beencorrelated with the status of patients with respect to muscle wasting(Gonzalez-Kadavid et al., Proc. Natl. Acad. Med., USA 95:14938-14943,1998, which is incorporated herein by reference). Thus, patients withAIDS, who also showed signs of cachexia as measured by loss of totalbody weight, had slightly increased serum levels of myostatinimmunoreactive material compared to either normal males without AIDS orto AIDS patients that did not have weight loss. However, theinterpretation of these results was complicated because the myostatinimmunoreactive material detected in the serum samples did not have themobility on SDS gels that was expected for authentic processedmyostatin.

As disclosed herein, myostatin not only affects muscle mass, but alsoaffects the overall metabolism of an organism. For example, myostatin isexpressed in adipose tissue, and myostatin deficient mice have adramatic reduction in fat accumulation as the animals age (see ExamplesII and III). Although no mechanism for myostatin action is proposedherein, the effect of myostatin can be direct effect of myostatin onadipose tissue, or can be an indirect effect caused by the lack ofmyostatin activity on skeletal muscle tissue. Regardless of themechanism, the overall anabolic effect on muscle tissue that results inresponse to decreased myostatin activity can alter the overallmetabolism of the organism and affect the storage of energy in the formof fat, as demonstrated by the introduction of a myostatin mutation intoan obese mouse strain (agouti lethal yellow (Ay) mice), which suppressedfat accumulation by five-fold (see Example 5). Abnormal glucosemetabolism also was partially suppressed in agouti mice containing themyostatin mutation. These results demonstrate that methods thatinhibition of myostatin can be used to treat or prevent metabolicdiseases such as obesity and type II diabetes.

The methods of the invention are useful, for example, for amelioratingthe severity of various pathologic conditions, including, for example,the cachexia associated with chronic diseases such as cancer (see Nortonet al., Crit. Rev. Oncol. Hematol. 7:289-327, 1987), as well asconditions such as type II diabetes, obesity, and other metabolicdisorders. As used herein, the term “pathologic condition” refers to adisorder that is characterized, at least in part, by an abnormal amount,development or metabolic activity of muscle or adipose tissue. Suchpathologic conditions, which include, for example, obesity; conditionsassociated with obesity, for example, atherosclerosis, hypertension, andmyocardial infarction; muscle wasting disorders such as musculardystrophy, neuromuscular diseases, cachexia, and anorexia; and metabolicdisorders such as type II diabetes, which generally, but notnecessarily, is associated with obesity, are particularly amenable totreatment using a method of the invention.

As used herein, the term “abnormal,” when used in reference to theamount, development or metabolic activity of muscle or adipose tissue,is used in a relative sense in comparison to an amount, development ormetabolic activity that a skilled clinician or other relevant artisanwould recognize as being normal or ideal. Such normal or ideal valuesare known to the clinician and are based on average values generallyobserved or desired in a healthy individual in a correspondingpopulation. For example, the clinician would know that obesity isassociated with a body weight that is about twenty percent above an“ideal” weight range for a person of a particular height and body type.However, the clinician would recognize that a body builder is notnecessarily obese simply by virtue of having a body weight that istwenty percent or more above the weight expected for a person of thesame height and body type in an otherwise corresponding population.Similarly, the artisan would know that a patient presenting with whatappears to abnormally decreased muscle activity could be identified ashaving abnormal muscle development, for example, by subjecting thepatient to various strength tests and comparing the results with thoseexpected for an average healthy individual in a correspondingpopulation.

A method of the invention can ameliorate the severity of a pathologiccondition that is characterized, at least in part, by an abnormalamount, development or metabolic activity in muscle or adipose tissue,by modulating myostatin signal transduction in a muscle or adiposetissue cell associated with the etiology of the condition. As usedherein, the term “ameliorate,” when used in reference to the severity ofa pathologic condition, means that signs or symptoms associated with thecondition are lessened. The signs or symptoms to be monitored will becharacteristic of a particular pathologic condition and will be wellknown to skilled clinician, as will the methods for monitoring the signsand conditions. For example, where the pathologic condition is type IIdiabetes, the skilled clinician can monitor the glucose levels, glucoseclearance rates, and the like in the subject. Where the pathologiccondition is obesity or cachexia, the clinician can simply monitor thesubject's body weight.

The agent to be administered to the subject is administered underconditions that facilitate contact of the agent with the target celland, if appropriate, entry into the cell. Entry of a polynucleotideagent into a cell, for example, can be facilitated by incorporating thepolynucleotide into a viral vector that can infect the cells. If a viralvector specific for the cell type is not available, the vector can bemodified to express a receptor (or ligand) specific for a ligand (orreceptor) expressed on the target cell, or can be encapsulated within aliposome, which also can be modified to include such a ligand (orreceptor). A peptide agent can be introduced into a cell by variousmethods, including, for example, by engineering the peptide to contain aprotein transduction domain such as the human immunodeficiency virus TATprotein transduction domain, which can facilitate translocation of thepeptide into the cell (see Schwarze et al., supra, 1999; Derossi et al.,supra, 1996).

The presence of the agent in the target cell can be identified directly,for example, by operatively linking a detectable label to the agent, byusing an antibody specific for the agent, particularly a peptide agent,or by detecting a downstream effect due to the agent, for example,decreased phosphorylation of an Smad polypeptide in the cell. An agentcan be labeled so as to be detectable using methods well known in theart (Hermanson, “Bioconjugate Techniques” (Academic Press 1996), whichis incorporated herein by reference; see, also, Harlow and Lane, supra,1988). For example, a peptide or polynucleotide agent can be labeledwith various detectable moieties including a radiolabel, an enzyme suchas alkaline phosphatase, biotin, a fluorochrome, and the like. Where theagent is contained in a kit, the reagents for labeling the agent alsocan be included in the kit, or the reagents can be purchased separatelyfrom a commercial source.

An agent useful in a method of the invention can be administered to thesite of the pathologic condition, or can be administered by any methodthat provides the target cells with the polynucleotide or peptide. Asused herein, the term “target cells” means muscle cells or adipocytesthat are to be contacted with the agent. For administration to a livingsubject, the agent generally is formulated in a pharmaceuticalcomposition suitable for administration to the subject. Thus, theinvention provides pharmaceutical compositions containing an agent,which is useful for modulating myostatin signal transduction in a cell,in a pharmaceutically acceptable carrier. As such, the agents are usefulas medicaments for treating a subject suffering from a pathologicalcondition as defined herein.

Pharmaceutically acceptable carriers are well known in the art andinclude, for example, aqueous solutions such as water or physiologicallybuffered saline or other solvents or vehicles such as glycols, glycerol,oils such as olive oil or injectable organic esters. A pharmaceuticallyacceptable carrier can contain physiologically acceptable compounds thatact, for example, to stabilize or to increase the absorption of theconjugate. Such physiologically acceptable compounds include, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients. Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the physico-chemical characteristics of thetherapeutic agent and on the route of administration of the composition,which can be, for example, orally or parenterally such as intravenously,and by injection, intubation, or other such method known in the art. Thepharmaceutical composition also can contain a second reagent such as adiagnostic reagent, nutritional substance, toxin, or therapeutic agent,for example, a cancer chemotherapeutic agent.

The agent can be incorporated within an encapsulating material such asinto an oil-in-water emulsion, a microemulsion, micelle, mixed micelle,liposome, microsphere or other polymer matrix (see, for example,Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla.1984); Fraley, et al., Trends Biochem. Sci., 6:77 (1981), each of whichis incorporated herein by reference). Liposomes, for example, whichconsist of phospholipids or other lipids, are nontoxic, physiologicallyacceptable and metabolizable carriers that are relatively simple to makeand administer. “Stealth” liposomes (see, for example, U.S. Pat. Nos.5,882,679; 5,395,619; and 5,225,212, each of which is incorporatedherein by reference) are an example of such encapsulating materialsparticularly useful for preparing a pharmaceutical composition usefulfor practicing a method of the invention, and other “masked” liposomessimilarly can be used, such liposomes extending the time that thetherapeutic agent remain in the circulation. Cationic liposomes, forexample, also can be modified with specific receptors or ligands(Morishita et al., J. Clin. Invest., 91:2580-2585 (1993), which isincorporated herein by reference). In addition, a polynucleotide agentcan be introduced into a cell using, for example, adenovirus-polylysineDNA complexes (see, for example, Michael et al., J. Biol. Chem.268:6866-6869 (1993), which is incorporated herein by reference).

The route of administration of a pharmaceutical composition containingan agent that alters myostatin signal transduction will depend, in part,on the chemical structure of the molecule. Polypeptides andpolynucleotides, for example, are not particularly useful whenadministered orally because they can be degraded in the digestive tract.However, methods for chemically modifying polypeptides, for example, torender them less susceptible to degradation by endogenous proteases ormore absorbable through the alimentary tract are well known (see, forexample, Blondelle et al., supra, 1995; Ecker and Crook, supra, 1995).In addition, a peptide agent can be prepared using D-amino acids, or cancontain one or more domains based on peptidomimetics, which are organicmolecules that mimic the structure of peptide domain; or based on apeptoid such as a vinylogous peptoid.

A pharmaceutical composition as disclosed herein can be administered toan individual by various routes including, for example, orally orparenterally, such as intravenously, intramuscularly, subcutaneously,intraorbitally, intracapsularly, intraperitoneally, intrarectally,intracisternally or by passive or facilitated absorption through theskin using, for example, a skin patch or transdermal iontophoresis,respectively. Furthermore, the pharmaceutical composition can beadministered by injection, intubation, orally or topically, the latterof which can be passive, for example, by direct application of anointment, or active, for example, using a nasal spray or inhalant, inwhich case one component of the composition is an appropriatepropellant. A pharmaceutical composition also can be administered to thesite of a pathologic condition, for example, intravenously orintra-arterially into a blood vessel supplying a tumor.

The total amount of an agent to be administered in practicing a methodof the invention can be administered to a subject as a single dose,either as a bolus or by infusion over a relatively short period of time,or can be administered using a fractionated treatment protocol, in whichmultiple doses are administered over a prolonged period of time. Oneskilled in the art would know that the amount of the pharmaceuticalcomposition to treat a pathologic condition in a subject depends on manyfactors including the age and general health of the subject as well asthe route of administration and the number of treatments to beadministered. In view of these factors, the skilled artisan would adjustthe particular dose as necessary. In general, the formulation of thepharmaceutical composition and the routes and frequency ofadministration are determined, initially, using Phase I and Phase IIclinical trials.

The pharmaceutical composition can be formulated for oral formulation,such as a tablet, or a solution or suspension form; or can comprise anadmixture with an organic or inorganic carrier or excipient suitable forenteral or parenteral applications, and can be compounded, for example,with the usual non-toxic, pharmaceutically acceptable carriers fortablets, pellets, capsules, suppositories, solutions, emulsions,suspensions, or other form suitable for use. The carriers, in additionto those disclosed above, can include glucose, lactose, mannose, gumacacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc,corn starch, keratin, colloidal silica, potato starch, urea, mediumchain length triglycerides, dextrans, and other carriers suitable foruse in manufacturing preparations, in solid, semisolid, or liquid form.In addition auxiliary, stabilizing, thickening or coloring agents andperfumes can be used, for example a stabilizing dry agent such astriulose (see, for example, U.S. Pat. No. 5,314,695).

The present invention also provides a method of modulating the growth ofmuscle tissue or adipose tissue in a subject. As disclosed herein, GDFreceptors such as Act RIIA and Act RIIB are involved in mediating theeffects of a GDF such as myostatin, which is involved in muscle tissueand adipose tissue formation. Thus, in one embodiment, a method ofmodulating the growth of muscle tissue or adipose tissue includesaffecting signal transduction from a GDF receptor, such as an activinreceptor, e.g., Act RIIA or Act RIIB. Such a method can be performed bycontacting cells of the tissue, or expressing in the cells, a mutant GDFreceptor, which has dominant negative activity, constitutive activity,or the like.

In another embodiment, a method of modulating the growth of muscletissue or adipose tissue in an organism is performed by administering tothe organism an agent that affects myostatin signal transduction.Preferably, the agent is or encodes a myostatin prodomain or a mutantpromyostatin polypeptide, either of which can include a myostatin signalpeptide. As used herein, the term “growth” is used in a relative sensein referring to the mass of muscle tissue or mass of adipose tissue inan organism that has been subjected to a method of the invention ascompared to a corresponding organism that has not been subjected to amethod of the invention. Thus, where a method of the invention isperformed such that myostatin signal transduction has been reduced orinhibited, it will be recognized that the growth of muscle tissue in theorganism would result in an increased muscle mass in the organism ascompared to the muscle mass of a corresponding organism (or populationof organisms) in which myostatin signal transduction had not been soeffected.

A method of the invention can be useful for increasing the muscle massor reducing the fat content of an organism or both. For example, wheresuch a method is performed on an organism that is useful as a foodsource, the protein content of the food can be increased, thecholesterol level can be decreased, and the quality of the foodstuff canbe improved. A method of the invention also can be useful for decreasingthe growth of muscle tissue in an organism, for example, an organismthat is detrimental to an environment, such that the organism is lessable to compete in the environment. Thus, a method of the invention canbe performed on any eukaryotic organism that expresses myostatin,including a vertebrate organism, for example, mammalian, avian orpiscine organism, or can be an invertebrate organism, for example, amollusk, echinoderm, gastropod or cephalopod.

The agent can be any agent that alters myostatin signal transduction, asdisclosed herein, and can be administered to the organism in anyconvenient manner. For example, where the organism to be treated arefish, shrimp, scallops, or the like, which are grown in aquaculture, theagent can be added to the water in which the organisms are maintained orcan be included in their food, particularly where the agent is a solublepeptide or a small organic molecule.

Where the agent used in a method of the invention is a polynucleotidethat encodes a peptide agent, an antisense agent, or the like, germcells of a non-human organism containing the polynucleotide can beselected and transgenic organisms expressing the agent can be produced.Preferably, the polynucleotide is under the control of an inducibleregulatory element, such that the agent encoded by the polynucleotidecan be expressed at a time and for a duration as desired. Accordingly,the present invention provides transgenic nonhuman organisms, as well asfood products produced by these organisms. Such food products haveincreased nutritional value because of the increase in muscle tissue.The transgenic non-human animals can be any species as disclosed herein,including vertebrate organisms such as cattle, pigs, sheep, chicken,turkey and fish, and invertebrate species such as shrimp, lobster,crabs, squid, oysters and abalone.

The regulation of TGF-θ family members and their specific interactionswith cell surface receptors are beginning to be elucidated. Thus,coexpression of the prodomain of a TGF-θ family member with a matureregion of another member of the TGF-θ family is associated withintracellular dimerization and secretion of biologically activehomodimers occur (Gray et al., Science 247:1328, 1990). For example, useof the BMP-2 prodomain with the BMP-4 mature region led to dramaticallyimproved expression of mature BMP-4 (Hammonds et al., (Mol. Endocrinol.5:149, 1991). For most of the family members that have been studied, thehomodimeric species are biologically active, whereas for other familymembers such as the inhibins (Ling et al., Nature 321:779, 1986) and theTGF-θs (Cheifetz et al., Cell 48:409, 1987), heterodimers also have beendetected and appear to have different biological properties than therespective homodimers.

Receptor-ligand interaction studies have revealed a great deal ofinformation as to how cells respond to external stimuli, and have led tothe development of therapeutically important compounds such aserythropoietin, the colony stimulating factors, and PDGF. Thus,continual efforts have been made at identifying the receptors thatmediate the action of the TGF-θ family members. As disclosed herein,myostatin specifically interacts with an activin type II receptor. Theidentification of this interaction provides targets for identifyingantagonists and agonists useful for agricultural and human therapeuticpurposes, for example, for treating in various pathological conditionssuch as obesity, type II diabetes, and cachexia. The identification ofthis specific interaction also provides a means to identify othermyostatin receptors, as well as the specific receptors of other growthdifferentiation factors. Accordingly, the present invention provides GDFreceptors, which specifically interact with a GDF or combination ofGDFs, for example, with myostatin, GDF-11, or both.

A GDF receptor of the invention is exemplified herein by a myostatinreceptor, particularly an activin type II receptor, which specificallyinteracts with myostatin and with GDF-11. However, myostatin receptorsthat specifically interact with myostatin, but not GDF-11, also areencompassed within the present invention, as are GDF-11 receptors thatspecifically interact with GDF-11 but not myostatin, and the like. Forconvenience of discussion, the receptors of the invention are referredto herein generally as a “GDF receptor” and are exemplified by amyostatin receptor, which is a receptor that specifically interacts atleast with myostatin. As such, while reference is made generally to aspecific interaction of myostatin with a myostatin receptor, it will berecognized that the present disclosure more broadly encompasses any GDFreceptor, including a GDF-11 receptor, which specifically interacts atleast with GDF-11.

A recombinant cell line that expresses a GDF receptor polypeptide alsois provided, as are antibodies that specifically bind the receptor,substantially purified polynucleotides that encode the receptor, andsubstantially purified GDF receptor polypeptides. Peptide portions of aGDF receptor also are provided, including, for example, solubleextracellular domains of a GDF receptor such as a myostatin receptor,which, as disclosed herein, can alter the specific interaction ofmyostatin with a cellular myostatin receptor; a constitutively activeintracellular kinase domain of a GDF receptor, which can induce,stimulate or otherwise maintain GDF signal transduction in a cell; orother truncated portion of a GDF receptor having an ability to modulatemyostatin or other GDF signal. transduction.

The invention also provides methods for identifying a GDF receptorpolypeptide, including methods of screening genomic or cDNA libraries,which can be expression libraries, using nucleotide probes or antibodyprobes; methods of screening cells that are responsive to and,therefore, express the receptor, using, for example, a GDF such asmyostatin or a functional peptide portion thereof; two hybrid assays, asdescribed above, using, for example, the GDF peptide as a component ofone hybrid and peptides expressed from a cDNA library, which is preparedfrom cells expressing a receptor for the GDF, as components of thesecond hybrids, and the like.

As described above, agents that specifically interact with a GDFreceptor, for example, a myostatin receptor such as Act RIIB can beidentified by using the receptor to screen for such agents. Conversely,an agent that has been identified as having the ability to specificallyinteract with a myostatin receptor such as the Act RIIB receptor, can beused to screen for additional myostatin receptors or other GDFreceptors. Such a method can include incubating components such as theagent (or myostatin or other GDF) and a cell expressing a GDF receptor,which can be a truncated membrane bound receptor or a soluble receptor,under conditions sufficient to allow the agent (or GDF) to interactspecifically with the receptor; measuring the agent (or GDF) bound tothe receptor; and isolating the receptor. A method of molecular modelingas described above also can be useful as a screening method foridentifying a GDF receptor, or a functional peptide portion thereof.

Non-human transgenic animals that have a phenotype characterized byexpression of a GDF receptor also are provided, the phenotype beingconferred by a transgene contained in the somatic and germ cells of theanimal. The transgene comprises a polynucleotide encoding the GDFreceptor, for example, myostatin receptor, polypeptide. Methods ofproducing such transgenic animals are disclosed herein or otherwiseknown in the art.

The present invention provides a substantially purified polynucleotideencoding all or a peptide portion of a GDF receptor. Although a GDFreceptor is exemplified herein by an activin type II receptor,polynucleotides encoding activin type II receptors previously have beendescribed (U.S. Pat. No. 5,885,794). Thus, it should be recognized thatsuch activin type II receptors are not encompassed within the presentinvention (Massague, supra, 1998; Heldin et al., supra, 1997).Similarly, activin type I receptors, including Act RIB; TGF-θ receptors,including TGF-θ RI and TGF-θ RII; and BMP receptors, including BMP MA,BMP RIB and BMP RII, have been described and are well known in the art(Massague, supra, 1998; Heldin et al., supra, 1997) and, therefore, arenot encompassed within the GDF receptors of the invention.

A polynucleotide of the invention can encode a polypeptide having amyostatin receptor activity, for example, myostatin binding activity, orcan encode a mutant myostatin receptor, for example, a mutant myostatinreceptor having a mutation in a kinase domain, such that the mutant actsas a dominant negative myostatin receptor (see above). Thus, apolynucleotide of the invention can be a naturally occurring, synthetic,or intentionally manipulated polynucleotide. For example, portions ofthe mRNA sequence can be altered due to alternate RNA splicing patternsor the use of alternate promoters for RNA transcription. As anotherexample, the polynucleotide can be subjected to site directedmutagenesis. The polynucleotide also can be antisense nucleotidesequence. GDF receptor polynucleotides of the invention includesequences that are degenerate as a result of the genetic code. There are20 natural amino acids, most of which are specified by more than onecodon. Therefore, all degenerate nucleotide sequences are includedwithin the invention, provided the amino acid sequence of the GDFreceptor polypeptide encoded by the polynucleotide is functionallyunchanged. Also included are nucleotide sequences that encode myostatinreceptor polypeptides.

Oligonucleotide portions of a polynucleotide encoding a GDF receptor ofthe invention also are encompassed within the present invention. Sucholigonucleotides generally are at least about 15 bases in length, whichis sufficient to permit the oligonucleotide to selectively hybridize toa polynucleotide encoding the receptor, and can be at least about 18nucleotides or 21 nucleotides or more in length. As used herein, theterm “selective hybridization” or “selectively hybridize” refers tohybridization under moderately stringent or highly stringentphysiological conditions, which can distinguish related nucleotidesequences from unrelated nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (for example, relativeGC:AT content), and nucleic acid type, i.e., whether the oligonucleotideor the target nucleic acid sequence is DNA or RNA, can be considered inselecting hybridization conditions. An additional consideration iswhether one of the nucleic acids is immobilized, for example, on afilter. Methods for selecting appropriate stringency conditions can bedetermined empirically or estimated using various formulas, and are wellknown in the art (see, for example, Sambrook et al., supra, 1989).

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and0.1×SSC at about 68° C. (high stringency conditions). Washing can becarried out using only one of these conditions, for example, highstringency conditions, or each of the conditions can be used, forexample, for 10 to 15 minutes each, in the order listed above, repeatingany or all of the steps listed.

A GDF receptor-encoding polynucleotide of the invention can be obtainedby any of several methods. For example, the polynucleotide can beisolated using hybridization or computer based techniques, as are wellknown in the art. These methods include, but are not limited to, 1)hybridization of genomic or cDNA libraries with probes to detecthomologous nucleotide sequences; 2) antibody screening of expressionlibraries to detect cloned DNA fragments with shared structuralfeatures; 3) polymerase chain reaction (PCR) on genomic DNA or cDNAusing primers capable of annealing to the DNA sequence of interest; 4)computer searches of sequence databases for similar sequences (seeabove); 5) differential screening of a subtracted DNA library; and 6)two hybrid assays using, for example, a mature GDF peptide in one of thehybrids.

In view of the present disclosure that an activin receptor specificallyinteracts with myostatin, oligonucleotide probes can be designed basedon the sequence encoding an activin receptor, for example, a sequenceencoding the extracellular domain, which binds myostatin, and used toscreen a library prepared from cells such as muscle cells or adipocytes,which are responsive to myostatin, thus facilitating identification of apolynucleotide encoding a myostatin receptor. Selected clones can befurther screened, for example, by subcloning the inserts into anexpression vector and, following expression of the cloned sequences,screening the expressed polypeptides using myostatin.

A polynucleotide of the invention, for example, a polynucleotideencoding a myostatin receptor, can be derived from a vertebrate species,including a mammalian, avian, or piscine species, or from aninvertebrate species. Screening procedures that rely on nucleic acidhybridization allow the isolation any gene sequence from any organism,provided the appropriate probe is available. Oligonucleotide probes thatcorrespond to a part of the sequence encoding the protein in questioncan be synthesized chemically. This requires that short, oligopeptidestretches of amino acid sequence are known. A polynucleotide sequenceencoding the receptor can be deduced from the genetic code, taking intoaccount the degeneracy of the genetic code. Thus, mixed additionreactions can be performed when the sequence is degenerate. Thisincludes a heterogeneous mixture of denatured double stranded DNA. Forsuch screening, hybridization is preferably performed on either singlestranded DNA or denatured double stranded DNA. Hybridization isparticularly useful in the detection of cDNA clones derived from sourceswhere an extremely low amount of mRNA sequences relating to thepolypeptide of interest are present. Thus, by using stringenthybridization conditions directed to avoid nonspecific binding,autoradiographic visualization can be used to identify a specific cDNAclone by the hybridization of the target DNA to an oligonucleotide probein the mixture that is the complete complement of the target nucleicacid (Wallace et al., Nucl. Acid Res., 9:879, 1981, which isincorporated herein by reference). Alternatively, a subtractive librarycan be used, thereby eliminating nonspecific cDNA clones.

When the entire amino acid sequence of a desired polypeptide is notknown, the direct synthesis of DNA sequences is not possible and themethod of choice is the synthesis of cDNA sequences. Among the standardprocedures for isolating cDNA sequences of interest is the formation ofcDNA libraries prepared in plasmids or phage, wherein the libraries arederived from reverse transcription of mRNA that is abundant in donorcells having a high level of genetic expression. When used incombination with polymerase chain reaction technology, even rareexpression products can be cloned. Where significant portions of theamino acid sequence of the polypeptide are known, the production oflabeled single stranded or double stranded DNA or RNA probe sequencesduplicating a sequence putatively present in the target cDNA can beemployed in hybridization procedures carried out on cloned copies of thecDNA, which have been denatured into a single stranded form (Jay et al.,Nucl. Acid Res., 11:2325, 1983, which is incorporated herein byreference).

A cDNA expression library, such as a lambda gt11 library, can bescreened for GDF receptor peptides using an antibody specific for a GDFreceptor, for example, an anti-Act RIIB antibody. The antibody can bepolyclonal or monoclonal, and can be used to detect expression productindicative of the presence of a GDF receptor cDNA. Such an expressionlibrary also can be screened with a GDF peptide, for example, withmyostatin, or a functional peptide portion thereof, to identify a cloneencoding at least a portion of a myostatin binding domain of a myostatinreceptor.

Polynucleotides encoding mutant GDF receptors and mutant GDF receptorpolypeptides are also encompassed within the invention. An alteration ina polynucleotide encoding a GDF receptor can be an intragenic mutationsuch as point mutation, nonsense (STOP) mutation, missense mutation,splice site mutation or frameshift, or can be a heterozygous orhomozygous deletion, and can be a naturally occurring mutation or can beengineered using recombinant DNA methods, for example. Such alterationscan be detected using standard methods known to those of skill in theart, including, but not limited to, nucleotide sequence analysis,Southern blot analysis, a PCR based analysis such as multiplex PCR orsequence tagged sites (STS) analysis, or in situ hybridization analysis.GDF receptor polypeptides can be analyzed by standard SDS-PAGE,immunoprecipitation analysis, western blot analysis, or the like. MutantGDF receptors are exemplified by truncated GDF receptors, including asoluble extracellular domain, which can have the ability to specificallybind its cognate GDF, but lacks a kinase domain; an intracellular GDFreceptor kinase domain, which can exhibit constitutive kinase activity;as well as by GDF receptors that contain a point mutation, whichdisrupts the kinase activity of the receptor or the ligand bindingability of the receptor; and the like. Such GDF receptor mutants areuseful for modulating GDF signal transduction and, therefore, forpracticing various methods of the invention.

A polynucleotide encoding a GDF receptor can be expressed in vitro byintroducing the polynucleotide into a suitable host cell. “Host cells”can be any cells in which the particular vector can be propagated, and,where appropriate, in which a polynucleotide contained in the vector canbe expressed. The term “host cells” includes any progeny of an originalhost cell. It is understood that all progeny of the host cell may not beidentical to the parental cell due, for example, to mutations that occurduring replication. However, such progeny are included when the term“host cell” is used. Methods of obtaining a host cell that transientlyor stably contains an introduced polynucleotide of the invention arewell known in the art.

A GDF receptor polynucleotide of the invention can be inserted into avector, which can be a cloning vector or a recombinant expressionvector. The term “recombinant expression vector” refers to a plasmid,virus or other vehicle known in the art that has been manipulated byinsertion or incorporation of a polynucleotide, particularly, withrespect to the present invention, a polynucleotide encoding all or apeptide portion of a GDF receptor. Such expression vectors contain apromoter sequence, which facilitates the efficient transcription of theinserted genetic sequence of the host. The expression vector generallycontains an origin of replication, a promoter, as well as specific geneswhich allow phenotypic selection of the transformed cells. Vectorssuitable for use in the present invention include, but are not limitedto, the T7-based expression vector for expression in bacteria(Rosenberg, et al., Gene 56:125, 1987), the pMSXND expression vector forexpression in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521,1988) and baculovirus-derived vectors for expression in insect cells.The DNA segment can be present in the vector operably linked toregulatory elements, for example, a promoter, which can be a T7promoter, metallothionein I promoter, polyhedrin promoter, or otherpromoter as desired, particularly tissue specific promoters or induciblepromoters.

A polynucleotide sequence encoding a GDF receptor can be expressed ineither prokaryotes or eukaryotes. Hosts can include microbial, yeast,insect and mammalian organisms. Methods of expressing polynucleotideshaving eukaryotic or viral sequences in prokaryotes are well known inthe art, as are biologically functional viral and plasmid DNA vectorscapable of expression and replication in a host. Methods forconstructing an expression vector containing a polynucleotide of theinvention are well known, as are factors to be considered in selectingtranscriptional or translational control signals, including, forexample, whether the polynucleotide is to be expressed preferentially ina particular cell type or under particular conditions (see, for example,Sambrook et al., supra, 1989).

A variety of host cell/expression vector systems can be utilized toexpress a GDF receptor coding sequence, including, but not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectors; yeastcells transformed with recombinant yeast expression vectors; plant cellsystems infected with recombinant virus expression vectors such as acauliflower mosaic virus or tobacco mosaic virus, or transformed withrecombinant plasmid expression vector such as a Ti plasmid; insect cellsinfected with recombinant virus expression vectors such as abaculovirus; animal cell systems infected with recombinant virusexpression vectors such as a retrovirus, adenovirus or vaccinia virusvector; and transformed animal cell systems genetically engineered forstable expression. Where the expressed GDF receptor ispost-translationally modified, for example, by glycosylation, it can beparticularly advantageous to select a host cell/expression vector systemthat can effect the desired modification, for example, a mammalian hostcell/expression vector system.

Depending on the host cell/vector system utilized, any of a number ofsuitable transcription and translation elements, including constitutiveand inducible promoters, transcription enhancer elements, transcriptionterminators, and the like can be used in the expression vector (Bitteret al., Meth. Enzymol. 153:516-544, 1987). For example, when cloning inbacterial systems, inducible promoters such as pL of bacteriophage Σ,plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used.When cloning in mammalian cell systems, promoters derived from thegenome of mammalian cells, for example, a human or mouse metallothioneinpromoter, or from mammalian viruses, for example, a retrovirus longterminal repeat, an adenovirus late promoter or a vaccinia virus 7.5Kpromoter, can be used. Promoters produced by recombinant DNA orsynthetic techniques can also be used to provide for transcription ofthe inserted GDF receptors coding sequence.

In yeast cells, a number of vectors containing constitutive or induciblepromoters can be used (see Ausubel et al., supra, 1987, see chapter 13;Grant et al., Meth. Enzymol. 153:516-544, 1987; Glover, DNA Cloning Vol.II (IRL Press, 1986), see chapter 3; Bitter, Meth. Enzymol. 152:673-684,1987; see, also, The Molecular Biology of the Yeast Saccharomyces (Eds.,Strathern et al., Cold Spring Harbor Laboratory Press, 1982), Vols. Iand II). A constitutive yeast promoter such as ADH or LEU2 or aninducible promoter such as GAL can be used (Rothstein, DNA Cloning Vol.II (supra, 1986), chapter 3). Alternatively, vectors can be used whichpromote integration of foreign DNA sequences into the yeast chromosome.

Eukaryotic systems, particularly mammalian expression systems, allow forproper post-translational modifications of expressed mammalian proteins.Eukaryotic cells which possess the cellular machinery for properprocessing of the primary transcript, glycosylation, phosphorylation,and advantageously, plasma membrane insertion of the gene product can beused as host cells for the expression of a GDF receptor polypeptide, orfunctional peptide portion thereof.

Mammalian cell systems which utilize recombinant viruses or viralelements to direct expression can be engineered. For example, when usingadenovirus expression vectors, the GDF receptors coding sequence can beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. Alternatively,the vaccinia virus 7.5K promoter can be used (Mackett et al., Proc.Natl. Acad. Sci., USA 79:7415-7419, 1982; Mackett et al., J. Virol.49:857-864, 1984; Panicali et al., Proc. Natl. Acad. Sci., USA79:4927-4931, 1982). Particularly useful are bovine papilloma virusvectors, which can replicate as extrachromosomal elements (Sarver etal., Mol. Cell. Biol. 1:486, 1981). Shortly after entry of this DNA intomouse cells, the plasmid replicates to about 100 to 200 copies per cell.Transcription of the inserted cDNA does not require integration of theplasmid into the host cell chromosome, thereby yielding a high level ofexpression. These vectors can be used for stable expression by includinga selectable marker in the plasmid, such as, for example, the neo gene.Alternatively, the retroviral genome can be modified for use as a vectorcapable of introducing and directing the expression of the GDF receptorsgene in host cells (Cone and Mulligan, Proc. Natl. Acad. Sci., USA81:6349-6353, 1984). High level expression can also be achieved usinginducible promoters, including, but not limited to, the metallothioneinIIA promoter and heat shock promoters.

For long term, high yield production of recombinant proteins, stableexpression is preferred. Rather than using expression vectors whichcontain viral origins of replication, host cells can be transformed withthe GDF receptors cDNA controlled by appropriate expression controlelements such as promoter, enhancer, sequences, transcriptionterminators, and polyadenylation sites, and a selectable marker. Theselectable marker in the recombinant plasmid can confer resistance tothe selection, and allows cells to stably integrate the plasmid intotheir chromosomes and grow to form foci, which, in turn can be clonedand expanded into cell lines. For example, following the introduction offoreign DNA, engineered cells can be allowed to grow for 1 to 2 days inan enriched media, and then are switched to a selective media. A numberof selection systems can be used, including, but not limited to, theherpes simplex virus thymidine kinase (Wigler et al., Cell 11:223,1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski, Proc. Natl. Acad. Sci., USA 48:2026, 1982), and adeninephosphoribosyltransferase (Lowy, et al., Cell 22:817, 1980) genes can beemployed in tk−, hgprt- or aprt-cells respectively. Also, antimetaboliteresistance can be used as the basis of selection for dhfr, which confersresistance to methotrexate (Wigler, et al., Proc. Natl. Acad. Sci. USA77:3567, 1980; O'Hare et al., Proc. Natl. Acad. Sci., USA 78: 1527,1981); gpt, which confers resistance to mycophenolic acid (Mulligan andBerg, Proc. Natl. Acad. Sci., USA 78:2072, 1981); neo, which confersresistance to the aminoglycoside G-418 (Colberre-Garapin et al., J. Mol.Biol. 150:1, 1981); and hygro, which confers resistance to hygromycin(Santerre et al., Gene 30:147, 1984) genes. Additional selectable genes,including trpB, which allows cells to utilize indole in place oftryptophan; hisD, which allows cells to utilize histinol in place ofhistidine (Hartman and Mulligan, Proc. Natl. Acad. Sci., USA 85:8047,1988); and ODC (ornithine decarboxylase) which confers resistance to theornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO(McConlogue, Curr. Comm. Mol. Biol. (Cold Spring Harbor LaboratoryPress, 1987), also have been described.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the GDF receptors of theinvention, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein. (Gluzman, Eukaryotic ViralVectors (Cold Spring Harbor Laboratory Press, 1982)).

The invention also provides stable recombinant cell lines, the cells ofwhich express GDF receptor polypeptides and contain DNA that encodes GDFreceptors. Suitable cell types include, but are not limited to, NIH 3T3cells (murine), C2C12 cells, L6 cells, and P19 cells. C2C12 and L6myoblasts differentiate spontaneously in culture and form myotubesdepending on the particular growth conditions (Yaffe and Saxel, Nature270:725-727, 1977; Yaffe, Proc. Natl. Acad. Sci., USA 61:477-483, 1968).P19 is an embryonal carcinoma cell line. Such cells are described, forexample, in the Cell Line Catalog of the American Type CultureCollection (ATCC). These cells can be stably transformed using wellknown methods (see, for example, Ausubel et al., supra, 1995, seesections 9.5.1-9.5.6).

A GDF receptor can be expressed from a recombinant polynucleotide of theinvention using inducible or constitutive regulatory elements, asdescribed herein. The desired protein encoding sequence and an operablylinked promoter can be introduced into a recipient cell either as anon-replicating DNA (or RNA) molecule, which can either be a linearmolecule or a covalently closed circular molecule. Expression of thedesired molecule can occur due to transient expression of the introducedsequence, or the polynucleotide can be stably maintained in the cell,for example, by integration into a host cell chromosome, thus allowing amore permanent expression. Accordingly, the cells can be stably ortransiently transformed (transfected) cells.

An example of a vector that can be employed is one which is capable ofintegrating the desired gene sequences into the host cell chromosome.Cells which have stably integrated the introduced DNA into theirchromosomes can be selected by also introducing one or more markerswhich allow for selection of host cells which contain the expressionvector. The marker can complement an auxotrophy in the host such asleu2, or ura3, which are common yeast auxotrophic markers; can confer abiocide resistance, for example, to an antibiotic or to heavy metal ionssuch as copper, or the like. The selectable marker gene can either bedirectly linked to the DNA gene sequences to be expressed, or can beintroduced into the same cell by cotransfection.

The introduced sequence can be incorporated into a plasmid or viralvector capable of autonomous replication in the recipient host. Any of avariety of vectors can be employed for this purpose. Factors ofimportance in selecting a particular plasmid or viral vector include theease with which recipient cells that contain the vector can berecognized and selected from those cells that do not contain the vector;the number of copies of the vector desired in a particular host cell;and whether it is desirable to be able to “shuttle” the vector betweenhost cells of different species.

For a mammalian host, several vector systems are available forexpression. One class of vectors utilizes DNA elements that provideautonomously replicating extra-chromosomal plasmids derived from animalviruses, for example, a bovine papilloma virus, polyoma virus,adenovirus, or SV40 virus. A second class of vectors includes vacciniavirus expression vectors. A third class of vectors relies upon theintegration of the desired gene sequences into the host chromosome.Cells that have stably integrated the introduced DNA into theirchromosomes can be selected by also introducing one or more markersgenes (as described above), which allow selection of host cells thatcontain the expression vector. The selectable marker gene can bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransfection. Additional elements can be included toprovide for optimal synthesis of an encoded mRNA or peptide, including,for example, splice signals, transcription promoters or enhancers, andtranscription or translation termination signals. cDNA expressionvectors incorporating appropriate regulatory elements are well known inthe art (see, for example, Okayama, Mol. Cell. Biol. 3:280, 1983).

Once the vector or DNA sequence containing the construct has beenprepared for expression, the DNA construct can be introduced into anappropriate host. Various methods can be used for introducing thepolynucleotide into a cell, including, for example, methods oftransfection or transformation such as protoplast fusion, calciumphosphate precipitation, and electroporation or other conventionaltechniques, for example, infection where the vector is a viral vector.

The invention also provides transgenic animals, which have cells thatexpress a recombinant GDF receptor. Such transgenic animals can beselected to have decreased fat content or increased muscle mass, orboth, and, therefore, can be useful as a source of food products withhigh muscle and protein content, and reduced fat and cholesterolcontent. The animals have been altered chromosomally in their germ cellsand somatic cells such that the production of a GDF, particularlymyostatin, is maintained at a normal” level, but the myostatin receptoris produced in reduced amounts, or is completely disrupted, resulting inthe cells in the animals having a decreased ability to bind myostatinand, consequently, having greater than normal levels of muscle tissue,preferably without increased fat or cholesterol levels. Accordingly, thepresent invention also includes food products provided by the animals.Such food products have increased nutritional value because of theincrease in muscle tissue. The transgenic non-human animals of theinvention include bovine, porcine, ovine and avian animals, as well asother vertebrates, and further includes transgenic invertebrates.

The invention also provides a method of producing animal food productshaving increased muscle content. Such a method can include modifying thegenetic makeup of the germ cells of a pronuclear embryo of the animal,implanting the embryo into the oviduct of a pseudopregnant female,thereby allowing the embryo to mature to full term progeny, testing theprogeny for presence of the transgene to identify transgene positiveprogeny, crossbreeding transgene positive progeny to obtain furthertransgene positive progeny, and processing the progeny to obtain afoodstuff. The modification of the germ cell comprises altering thegenetic composition so as to reduce or inhibit the expression of thenaturally occurring gene encoding for production of a myostatin receptorprotein. For example, the transgene can comprise an antisense moleculethat is specific for a polynucleotide encoding a myostatin receptor; cancomprise a non-functional sequence that replaces or intervenes in theendogenous myostatin receptor gene or the transgene; or can encode amyostatin receptor antagonist, for example, a dominant negativemyostatin receptor such as a dominant negative Act RIIB.

As used herein, the term “animal” refers to any bird, fish or mammal,except a human, and includes any stage of development, includingembryonic and fetal stages. Farm animals such as pigs, goats, sheep,cows, horses, rabbits and the like; rodents such as mice; and domesticpets such as cats and dogs are included within the meaning of the term“animal.” In addition, the term “organism” is used herein to includeanimals as described above, as well as other eukaryotes, including, forexample, other vertebrates such as reptiles and amphibians, as well asinvertebrates as described above.

As used herein, the term “transgenic,” when used in reference to ananimal or an organism, means that cells of the animal or organism havebeen genetically manipulated to contain an exogenous polynucleotidesequence that is stably maintained with the cells. The manipulation canbe, for example, microinjection of a polynucleotide or infection with arecombinant virus containing the polynucleotide. Thus, the term“transgenic” is used herein to refer to animals (organisms) in which oneor more cells receive a recombinant polynucleotide, which can beintegrated into a chromosome in the cell, or can be maintained as anextrachromosomally replicating polynucleotide, such as might beengineered into a yeast artificial chromosome. The term “transgenicanimal” also includes a “germ cell line” transgenic animal. A germ cellline transgenic animal is a transgenic animal in which the geneticinformation has been taken up and incorporated into a germ line cell,therefore conferring the ability to transfer the information tooffspring. If such offspring in fact possess some or all of thatinformation, the offspring also are considered to be transgenic animals.The invention further encompasses transgenic organisms.

A transgenic organism can be any organism whose genome has been alteredby in vitro manipulation of an early stage embryo or a fertilized egg,or by any transgenic technology to induce a specific gene knock-out. Theterm “gene knock-out” refers to the targeted disruption of a gene in acell or in vivo that results in complete loss of function. A target genein a transgenic animal can be rendered nonfunctional by an insertiontargeted to the gene to be rendered nonfunctional, for example, byhomologous recombination, or by any other method for disrupting thefunction of a gene in a cell.

The transgene to be used in the practice of the subject invention can bea DNA sequence comprising a modified GDF receptors coding sequence.Preferably, the modified GDF receptor gene is one that is disrupted byhomologous targeting in embryonic stem cells. For example, the entiremature C-terminal region of the GDF receptors gene can be deleted (seeExample 13). Optionally, the disruption (or deletion) can be accompaniedby insertion of or replacement with another polynucleotide, for example,a nonfunctional GDF receptor sequence. A “knock-out” phenotype also canbe conferred by introducing or expressing an antisense GDF receptorpolynucleotide in a cell in the organism, or by expressing an antibodyor a dominant negative GDF receptor in the cells. Where appropriate,polynucleotides that encode proteins having GDF receptor activity, butthat differ in nucleotide sequence from a naturally occurring GDF genesequence due to the degeneracy of the genetic code, can be used herein,as can truncated forms, allelic variants and interspecies homologs.

The present invention also provides antibodies that specifically bind aGDF receptor, and that block GDF binding to the receptor. Suchantibodies can be useful, for example, for ameliorating a pathologiccondition such as a cell proliferative disorder associated with muscletissue.

A monoclonal antibody that binds specifically to a GDF receptor,particularly to a myostatin receptor, can increase the development ofskeletal muscles. In preferred embodiments of the claimed methods, a GDFreceptor monoclonal antibody, polypeptide, or polynucleotide isadministered to a patient suffering from a pathologic condition such asa muscle wasting disease, a neuromuscular disorder, muscle atrophy,aging, or the like. The GDF receptor antibody, particularly ananti-myostatin receptor antibody, can also be administered to a patientsuffering from a pathologic condition such as a muscular dystrophy,spinal cord injury, traumatic injury, congestive obstructive pulmonarydisease (COPD), AIDS or cachexia.

In a preferred embodiment, the anti-myostatin receptor antibody isadministered to a patient with muscle wasting disease or disorder byintravenous, intramuscular or subcutaneous injection; preferably, amonoclonal antibody is administered within a dose range between about0.1 Tg/kg to about 100 mg/kg; more preferably between about 1 Tg/kg to75 mg/kg; most preferably from about 10 mg/kg to 50 mg/kg. The antibodycan be administered, for example, by bolus injunction or by slowinfusion. Slow infusion over a period of 30 minutes to 2 hours ispreferred. The anti-myostatin receptor antibody, or other anti-GDFreceptor antibody, can be formulated in a formulation suitable foradministration to a patient. Such formulations are known in the art.

The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of the myostatinreceptor protein, for example, amount of tissue desired to be formed,the site of tissue damage, the condition of the damaged tissue, the sizeof a wound, type of damaged tissue, the patient's age, sex, and diet,the severity of any infection, time of administration and other clinicalfactors. The dosage can vary with the type of matrix used in thereconstitution and the types of agent, such as anti-myostatin receptorantibodies, to be used in the composition. Generally, systemic orinjectable administration, such as intravenous, intramuscular orsubcutaneous injection. Administration generally is initiated at a dosewhich is minimally effective, and the dose is increased over apreselected time course until a positive effect is observed.Subsequently, incremental increases in dosage are made limiting suchincremental increases to such levels that produce a correspondingincrease in effect, while taking into account any adverse affects thatcan appear. The addition of other known growth factors, such as IGF I(insulin like growth factor I), human, bovine, or chicken growthhormone, which can aid in increasing muscle mass, to the finalcomposition, can also affect the dosage. In the embodiment where ananti-myostatin receptor antibody is administered, the antibody isgenerally administered within a dose range of about 0.1 Tg/kg to about100 mg/kg.; more preferably between about 10 mg/kg to 50 mg/kg.

As used herein, the term “antibody” is used in its broadest sense toinclude polyclonal and monoclonal antibodies, as well as antigen bindingfragments of such antibodies. An antibody useful in a method of theinvention, or an antigen binding fragment thereof, is characterized, forexample, by having specific binding activity for an epitope of a GDFreceptor, for example, a myostatin receptor. In addition, as discussedabove, an antibody of the invention can be an antibody that specificallybinds a peptide portion of a promyostatin polypeptide, particularly amyostatin prodomain or functional peptide portion thereof. It will berecognized that the following methods, which exemplify the preparationand characterization of GDF receptor antibodies, further are applicableto the preparation and characterization of additional antibodies of theinvention, including antibodies that specifically bind a myostatinprodomain, antibodies that specifically bind a promyostatin polypeptideand reduce or inhibit proteolytic cleavage of the promyostatin tomyostatin, and the like.

The term “binds specifically” or “specific binding activity,” when usedin reference to an antibody means that an interaction of the antibodyand a particular epitope has a dissociation constant of at least about1×10-6, generally at least about 1×10-7, usually at least about 1×10-8,and particularly at least about 1×10-9 or 1×10-10 or less. As such, Fab,F(ab′)2, Fd and Fv fragments of an antibody that retain specific bindingactivity for an epitope of a GDF receptor, are included within thedefinition of an antibody. For purposes of the present invention, anantibody that reacts specifically with an epitope of a myostatinreceptor, for example, is considered to not substantially react with aTGF-θ receptor or a BMP receptor if the antibody has at least a two-foldgreater binding affinity, generally at least a five-fold greater bindingaffinity, and particularly at least a ten-fold greater binding affinityfor the myostatin receptor as compared to the TGF-θ or BMP receptor.

The term “antibody” as used herein includes naturally occurringantibodies as well as non-naturally occurring antibodies, including, forexample, single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof. Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains (see Huse et al., Science246:1275-1281 (1989), which is incorporated herein by reference). Theseand other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies are well known tothose skilled in the art (Winter and Harris, Immunol. Today 14:243-246,1993; Ward et al., Nature 341:544-546, 1989; Harlow and Lane,Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press,1988); Hilyard et al., Protein Engineering: A practical approach (IRLPress 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford UniversityPress 1995); each of which is incorporated herein by reference).

Antibodies that bind specifically with a GDF receptor can be raisedusing the receptor as an immunogen and removing antibodies thatcrossreact, for example, with a TGF-θ type I or type II receptor, withan activin receptor such as Act RIB, Act RIIA or Act RIIB, or a BMPreceptors such as BMP R11, BMP RIA and BMP RIB (see Massague, supra,1998). An antibody of the invention conveniently can be raised using apeptide portion of a myostatin receptor that is not present in a TGF-θ,activin, or BMP receptor. Similarly, an antibody that specifically bindsa myostatin prodomain can be raised using the prodomain, or a functionalpeptide portion thereof as the immunogen. Where such a peptide isnon-immunogenic, it can be made immunogenic by coupling the hapten to acarrier molecule such as bovine serum albumin (BSA) or keyhole limpethemocyanin (KLH), or by expressing the peptide portion as a fusionprotein. Various other carrier molecules and methods for coupling ahapten to a carrier molecule are well known in the art (see, forexample, by Harlow and Lane, supra, 1988).

If desired, a kit incorporating an antibody or other agent useful in amethod of the invention can be prepared. Such a kit can contain, inaddition to the agent, a pharmaceutical composition in which the agentcan be reconstituted for administration to a subject. The kit also cancontain, for example, reagents for detecting the antibody, or fordetecting specific binding of the antibody to a GDF receptor. Suchdetectable reagents useful for labeling or otherwise identifying theantibody are described herein and known in the art.

Methods for raising polyclonal antibodies, for example, in a rabbit,goat, mouse or other mammal, are well known in the art (see, forexample, Green et al., “Production of Polyclonal Antisera,” inImmunochemical Protocols (Manson, ed., Humana Press 1992), pages 1-5;Coligan et al., “Production of Polyclonal Antisera in Rabbits, Rats,Mice and Hamsters,” in Curr. Protocols Immunol. (1992), section 2.4.1;each or which is incorporated herein by reference). In addition,monoclonal antibodies can be obtained using methods that are well knownand routine in the art (Harlow and Lane, supra, 1988). For example,spleen cells from a mouse immunized with a myostatin receptor, or anepitopic fragment thereof, can be fused to an appropriate myeloma cellline such as SP/02 myeloma cells to produce hybridoma cells. Clonedhybridoma cell lines can be screened using labeled antigen to identifyclones that secrete monoclonal antibodies having the appropriatespecificity, and hybridomas expressing antibodies having a desirablespecificity and affinity can be isolated and utilized as a continuoussource of the antibodies. The antibodies can be further screened for theinability to bind specifically with the myostatin receptor. Suchantibodies are useful, for example, for preparing standardized kits forclinical use. A recombinant phage that expresses, for example, a singlechain anti-myostatin receptor antibody also provides an antibody thatcan used for preparing standardized kits.

Methods of preparing monoclonal antibodies well known (see, for example,Kohler and Milstein, Nature 256:495, 1975, which is incorporated hereinby reference; see, also, Coligan et al., supra, 1992, see sections2.5.1-2.6.7; Harlow and Lane, supra, 1988). Briefly, monoclonalantibodies can be obtained by injecting mice with a compositioncomprising an antigen, verifying the presence of antibody production byremoving a serum sample, removing the spleen to obtain B lymphocytes,fusing the B lymphocytes with myeloma cells to produce hybridomas,cloning the hybridomas, selecting positive clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well established techniques, including, forexample, affinity chromatography with Protein-A SEPHAROSE, sizeexclusion chromatography, and ion exchange chromatography (Coligan etal., supra, 1992, see sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3;see, also, Barnes et al., “Purification of Immunoglobulin G (IgG),” inMeth.: Molec. Biol. 10:79-104 (Humana Press 1992), which is incorporatedherein by reference). Methods of in vitro and in vivo multiplication ofmonoclonal antibodies is well known to those skilled in the art.Multiplication in vitro can be carried out in suitable culture mediasuch as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionallyreplenished by a mammalian serum such as fetal calf serum or traceelements and growth sustaining supplements such as normal mouseperitoneal exudate cells, spleen cells, bone marrow macrophages.Production in vitro provides relatively pure antibody preparations andallows scale-up to yield large amounts of the desired antibodies. Largescale hybridoma cultivation can be carried out by homogenous suspensionculture in an airlift reactor, in a continuous stirrer reactor, or inimmobilized or entrapped cell culture. Multiplication in vivo can becarried out by injecting cell clones into mammals histocompatible withthe parent cells, for example, syngeneic mice, to cause growth ofantibody producing tumors. Optionally, the animals are primed with ahydrocarbon, especially oils such as pristane (tetramethylpentadecane)prior to injection. After one to three weeks, the desired monoclonalantibody is recovered from the body fluid of the animal.

Therapeutic applications for antibodies disclosed herein are also partof the present invention. For example, antibodies of the presentinvention can also be derived from subhuman primate antibody. Generaltechniques for raising therapeutically useful antibodies in baboons canbe found, for example, in Goldenberg et al., International PatentPublication WO 91/11465 (1991); and Losman et al., Int. J. Cancer46:310, 1990, each of which is incorporated herein by reference.

A therapeutically useful anti-GDF receptor antibody also can be derivedfrom a “humanized” monoclonal antibody. Humanized monoclonal antibodiesare produced by transferring mouse complementarity determining regionsfrom heavy and light variable chains of the mouse immunoglobulin into ahuman variable domain, and then substituting human residues in theframework regions of the murine counterparts. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of murine constantregions. General techniques for cloning murine immunoglobulin variabledomains are known (see, for example, Orlandi et al., Proc. Natl. Acad.Sci., USA 86:3833, 1989, which is hereby incorporated in its entirety byreference). Techniques for producing humanized monoclonal antibodiesalso are known (see, for example, Jones et al., Nature 321:522, 1986;Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci., USA 89:4285,1992; Sandhu, Crit. Rev. Biotechnol. 12:437, 1992; and Singer et al., J.Immunol. 150:2844, 1993; each of which is incorporated herein byreference).

Antibodies of the invention also can be derived from human antibodyfragments isolated from a combinatorial immunoglobulin library (see, forexample, Barbas et al., METHODS: A Companion to Methods in Immunology2:119, 1991; Winter et al., Ann. Rev. Immunol. 12:433, 1994; each ofwhich is incorporated herein by reference). Cloning and expressionvectors that are useful for producing a human immunoglobulin phagelibrary can be obtained, for example, from STRATAGENE Cloning Systems(La Jolla, Calif.).

An antibody of the invention also can be derived from a human monoclonalantibody. Such antibodies are obtained from transgenic mice that havebeen “engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described, forexample, by Green et al., Nature Genet. 7:13, 1994; Lonberg et al.,Nature 368:856, 1994; and Taylor et al., Int. Immunol. 6:579, 1994; eachof which is incorporated herein by reference.

Antibody fragments of the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofDNA encoding the fragment. Antibody fragments can be obtained by pepsinor papain digestion of whole antibodies by conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)2. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab′ fragments and an Fc fragment directly (see, forexample, Goldenberg, U.S. Pat. No. 4,036,945 and U.S. Pat. No.4,331,647, each of which is incorporated by reference, and referencescontained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230.1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Meth. Enzymol.,1:422 (Academic Press 1967), each of which is incorporated herein byreference; see, also, Coligan et al., supra, 1992, see sections2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light/heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques can alsobe used, provided the fragments specifically bind to the antigen that isrecognized by the intact antibody. For example, Fv fragments comprise anassociation of VH and VL chains. This association can be noncovalent(Inbar et al., Proc. Natl. Acad. Sci., USA 69:2659, 1972).Alternatively, the variable chains can be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde(Sandhu, supra, 1992). Preferably, the Fv fragments comprise VH and VLchains connected by a peptide linker. These single-chain antigen bindingproteins (sFv) are prepared by constructing a structural gene comprisingDNA sequences encoding the VH and VL domains connected by anoligonucleotide. The structural gene is inserted into an expressionvector, which is subsequently introduced into a host cell such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingsFvs are described, for example, by Whitlow et al., Methods: A Companionto Methods in Enzymology 2:97, 1991; Bird et al., Science 242:423-426,1988; Ladner et al., U.S. Pat. No. 4,946,778; Pack et al.,Bio/Technology 11:1271-1277, 1993; each of which is incorporated hereinby reference; see, also Sandhu, supra, 1992.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106, 1991, which isincorporated herein by reference).

The invention also provides a method for identifying a GDF receptorpolypeptide. Such a method can be performed, for example, by incubatingcomponents comprising GDF polypeptide and a cell expressing a fulllength receptor or truncated receptor under conditions sufficient toallow the GDF to bind to the receptor; measuring the binding of the GDFpolypeptide to the receptor; and isolating the receptor. The GDF can beany of the known GDFs (e.g., GDF-1-16), and preferably is GDF-8(myostatin) or GDF-11. Methods of isolating the receptors are describedin more detail in the Examples section below. Accordingly, the inventionalso provides a substantially purified GDF receptor, as well as peptidesand peptide derivatives of a GDF receptor that have fewer amino acidresidues than a naturally occurring GDF receptor. Such peptides andpeptide derivatives are useful as research and diagnostic tools in thestudy of muscle wasting diseases and the development of more effectivetherapeutics.

The invention further provides GDF receptor variants. As used herein,the term “GDF receptors variant” means a molecule that simulates atleast part of the structure of GDF receptors. GDF receptor variants canbe useful in reducing or inhibiting GDF binding, thereby ameliorating apathologic condition as disclosed herein. Examples of GDF receptorvariants include, but are not limited to, truncated GDF receptors suchas a soluble extracellular domain of a GDF receptor; a dominant negativeGDF receptor such as a dominant negative Act RIIB receptor, which lackskinase activity; or other truncated or mutant GDF receptors.

The invention relates not only to peptides and peptide derivatives of anaturally-occurring GDF receptor, but also to GDF receptor variants,including mutants GDF receptors, and chemically synthesized derivativesof GDF receptors that specifically bind a GDF, for example, myostatin.For example, changes in the amino acid sequence of a GDF receptor arecontemplated in the present invention. GDF receptors can be altered bychanging the DNA encoding the protein. Preferably, only conservativeamino acid alterations are undertaken, using amino acids that have thesame or similar properties. Illustrative amino acid substitutionsinclude the changes of alanine to serine; arginine to lysine; asparagineto glutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparagine; glutamate to aspartate; glycine to proline;histidine to asparagine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine, glutamine, orglutamate; methionine to leucine or isoleucine; phenylalanine totyrosine, leucine or methionine; serine to threonine; threonine toserine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine;valine to isoleucine or leucine.

Variants useful for the present invention comprise analogs, homologs,muteins and mimetics of a GDF receptor that retain the ability tospecifically bind to their respective GDFs. In another embodiment,variant GDF receptors that have dominant negative activity also arecontemplated, regardless of whether the variant also interactsspecifically with its GDF. Peptides of the GDF receptors refer toportions of the amino acid sequence of GDF receptors that have theseabilities. The variants can be generated directly from GDF receptorsitself by chemical modification, by proteolytic enzyme digestion, or bycombinations thereof. Additionally, genetic engineering techniques, aswell as methods of synthesizing polypeptides directly from amino acidresidues, can be employed.

Peptides can be synthesized by such commonly used methods as t-BOC orFMOC protection of alpha-amino groups. Both methods involve stepwisesyntheses whereby a single amino acid is added at each step startingfrom the C terminus of the peptide (Coligan, et al., Current Protocolsin Immunology (Wiley Interscience, 1991), Unit 9, which is incorporatedherein by reference). Peptides of the invention can also be synthesizedby the well known solid phase peptide synthesis methods (Merrifield, J.Am. Chem. Soc., 85:2149, 1962; Stewart and Young, Solid Phase PeptidesSynthesis (Freeman, San Francisco, 1969), see pages 27-62, each of whichis incorporated herein by reference), using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.On completion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout ¼-1 hours at 0° C. After evaporation of the reagents, the peptidesare extracted from the polymer with 1% acetic acid solution which isthen lyophilized to yield the crude material. This can normally bepurified by such techniques as gel filtration on Sephadex G-15 using 5%acetic acid as a solvent. Lyophilization of appropriate fractions of thecolumn will yield the homogeneous peptide or peptide derivatives, whichcan then be characterized by such standard techniques as amino acidanalysis, thin layer chromatography, high performance liquidchromatography, ultraviolet absorption spectroscopy, molar rotation,solubility, and quantitated by the solid phase Edman degradation.

Non-peptide compounds that mimic the binding and function of GDFreceptors (“mimetics”) can be produced by the approach outlined bySaragovi et al. (Science 253: 792-95, 1991, which is incorporated hereinby reference). Mimetics are molecules which mimic elements of proteinsecondary structure (Johnson et al., “Peptide Turn Mimetics,” inBiotechnology and Pharmacy (Pezzuto et al., Eds.; Chapman and Hall, NewYork 1993), which is incorporated herein by reference). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions. For the purposes ofthe present invention, an appropriate mimetic can be considered to bethe equivalent of a GDF receptor.

Longer peptides can be produced by the “native chemical” ligationtechnique which links together peptides (Dawson et al., Science 266:776,1994, which is incorporated herein by reference). Variants can becreated by recombinant techniques employing genomic or cDNA cloningmethods. Site specific and region directed mutagenesis techniques can beemployed (Ausubel et al., supra, 1989 and 1990 to 1993 supplements), seevolume 1, chapter 8; Protein Engineering (Oxender and Fox eds., A. Liss,Inc., 1987)). In addition, linker scanning and PCR mediated techniquescan be employed for mutagenesis (Erlich, PCR Technology (Stockton Press1989); Ausubel et al., supra, 1989 to 1993). Protein sequencing,structure and modeling approaches for use with any of the abovetechniques are disclosed in the above cited references.

The present invention also provides GDF receptor-binding agents thatblock the specific binding of a GDF to its receptor. Such agents areuseful, for example, as research and diagnostic tools in the study ofmuscle wasting disorder as described above and as effectivetherapeutics, and can be identified using the methods as disclosedherein, for example, a molecular modeling method. In addition,pharmaceutical compositions comprising GDF receptor-binding agents canrepresent effective therapeutics. In the context of the invention, thephrase “GDF receptor-binding agent” denotes a naturally occurring ligandof a GDF receptor, for example, GDF-1 to GDF-16; a synthetic ligand ofGDF receptors, or an appropriate derivative of the natural or syntheticligands. The determination and isolation of ligands is well known in theart (Lerner, Trends Neurosci. 17:142-146, 1994, which is incorporatedherein by reference).

In yet another embodiment, the present invention relates to GDFreceptor-binding agents that interfere with binding between a GDFreceptor and a GDF. Such binding agents can interfere by competitiveinhibition, by non-competitive inhibition or by uncompetitiveinhibition. Interference with normal binding between GDF receptors andone or more GDF can result in a useful pharmacological effect.

The invention also provides a method for identifying a composition thatbinds to a GDF receptor. The method includes incubating componentscomprising the composition and a GDF receptor under conditionssufficient to allow the components to interact specifically, andmeasuring the binding of the composition to GDF receptors. Compositionsthat bind to GDF receptors include peptides, peptidomimetics,polypeptides, chemical compounds and biologic agents as described above.Incubating includes exposing the reactants to conditions that allowcontact between the test composition and GDF receptors, and provideconditions suitable for a specific interaction as would occur in vivo.Contacting can be in solution or in solid phase. The testligand/composition can optionally be a combinatorial library forscreening a plurality of compositions, as described above. Compositionsidentified in the method of the invention can be further evaluated,detected, cloned, sequenced, and the like, either in solution or afterbinding to a solid support, by any method usually applied to thedetection of a specific DNA sequence such as PCR, oligomer restriction(Saiki et al., Bio/Technology 3:1008-1012, 1985, which is incorporatedherein by reference), allele-specific oligonucleotide (ASO) probeanalysis (Conner et al., Proc. Natl. Acad. Sci., USA 80:278, 1983, whichis incorporated herein by reference), oligonucleotide ligation assays(OLAs) (Landegren et al., Science 241:1077 1988, which is incorporatedherein by reference), and the like (see Landegren et al., Science242:229-237, 1988. which is incorporated herein by reference).

To determine if a composition can functionally complex with the receptorprotein, induction of an exogenous gene can be monitored by monitoringchanges in the protein level of a protein encoded for by the exogenousgene, or any other method as disclosed herein. When a compositions isidentified that can induce transcription of the exogenous gene, it isconcluded that this composition can specifically bind to the receptorprotein coded for by the nucleic acid encoding the initial sample testcomposition.

Expression of the exogenous gene can be monitored by a functional assayor assay for a protein product, for example. The exogenous gene istherefore a gene that provides an assayable/measurable expressionproduct in order to allow detection of expression of the exogenous gene.Such exogenous genes include, but are not limited to, reporter genessuch as chloramphenicol acetyltransferase gene, an alkaline phosphatasegene, θ-galactosidase, a luciferase gene, a green fluorescent proteingene, guanine xanthine phosphoribosyltransferase, alkaline phosphatase,and antibiotic resistance genes such as neomycin phosphotransferase (seeabove).

Expression of the exogenous gene is indicative of a specific interactionof a composition and a GDF receptor; thus, the binding or blockingcomposition can be identified and isolated. The compositions of thepresent invention can be extracted and purified from the culture mediaor a cell by using known protein purification techniques commonlyemployed, including, for example, extraction, precipitation, ionexchange chromatography, affinity chromatography, gel filtration and thelike. Compositions can be isolated by affinity chromatography using themodified receptor protein extracellular domain bound to a column matrixor by heparin chromatography.

Also included in the screening method of the invention is combinatorialchemistry methods for identifying chemical compounds that bind to GDFreceptors, as described above. Thus, the screening method is also usefulfor identifying variants, binding or blocking agents, etc., whichfunctionally, if not physically (e.g., sterically) act as antagonists oragonists, as desired.

The following examples are intended to illustrate but not limit theinvention.

Example 1 Myostatin Acts in a Dose Dependent Manner

This example demonstrates that the activity of myostatin in inhibitingmuscle growth is dependent on the level of myostatin expression in vivo.

Myostatin is a negative regulator of skeletal muscle mass (McPherron etal., supra, 1997; McPherron and Lee, supra, 1997). Myostatin knock-outmice that were homozygous for a deletion of the myostatin gene had a25-30% increase in total body mass. An examination of the homozygousknock-out mice revealed that the increased muscle mass was due to abouta 100-200% increase in skeletal muscle mass throughout the body.

Mice that were heterozygous for the myostatin mutation also had anincrease in total body mass. However, the increase mass of theheterozygotes was less than that of the homozygotes, and wasstatistically significant in only one age and sex group among the manyexamined. In order to determine whether heterozygous mice have anintermediate phenotype between that of wild type mice and homozygousmyostatin knock-out mice, the analysis of muscle weights was extended tothe heterozygous mice. Individual muscles sampled from heterozygous miceweighed approximately 25-50% more than those of wild type mice. Theseresults demonstrate that mice that are heterozygous for deletion of amyostatin gene have a phenotype that is intermediate between that ofwild type mice and homozygous myostatin knock-out mice, and demonstratethat myostatin produces a dose-dependent effect in vivo.

These results indicate that the manipulation of myostatin activity canbe useful in treating muscle wasting diseases and other metabolicdisorders associated with myostatin activity. Furthermore, thedose-dependent effect of myostatin indicates that a therapeutic effectcan be obtained without achieving complete inhibition of myostatinactivity, thereby allowing for an adjustment of myostatin activity if,for example, a certain level of activity produces undesirable effects ina subject.

Example 2 Myostatin Effect Decreases with Age in Knock-Out Mice

This example demonstrates that a decreased difference in body weightbetween wild type mice and homozygous myostatin knock-out mice isassociated with a decline in muscle weight of the mutant mice.

Myostatin knock-out mice weighed approximately 25-30% more than wildtype mice at five months of age (McPherron et al., supra, 1997).However, this difference in total body weights became significantlysmaller or disappeared altogether as the animals aged. In order todetermine whether this effect was due to a relative loss of weight inthe knock-out mice due, for example, to muscle degeneration, or to arelatively greater weight gain by wild type mice, a detailed analysis ofmuscle weights was made as a function of age.

At all ages examined from 2 months to 17 months, the pectoralis muscleweighed significantly more in homozygous mutant mice than in wild typelittermates. The most dramatic difference was observed at 5 months, atwhich time the pectoralis weight was approximately 200% greater in themutant mice. Although the pectoralis weight declined slightly at olderages, the weight of this muscle in mutant mice remained greater thantwice that of wild type mice. This same basic trend was observed in allof the other muscles examined, including the triceps brachii, thequadriceps, the gastrocnemius and plantaris, and the tibialis. anterior.Similar trends were observed in both male and female mice. These resultsdemonstrate that the decreased difference in total body weights betweenmutant and wild type mice observed with aging is due to a slight declinein muscle weights in the mutant mice.

Example 3 Myostatin Affects Fat Accumulation in a Dose Dependent Manner

This example demonstrates that myostatin knock-out mice fail toaccumulate fat, and that the decrease in fat accumulation is associatedwith the level of myostatin expression in vivo.

Since the decline in muscle weights in myostatin mutants, asdemonstrated in Example 2, did not fully account for the observationthat the wild type animals eventually weighed about the same as themutant mice, the amount of fat accumulation in wild type and mutant micewas examined. The inguinal, epididymal and retroperitoneal fat pads inmale mice were examined. There was no difference in the weights of anyof these fat pads between wild type and mutant mice at two months ofage. By 5 to 6 months of age, wild type and heterozygous knock-out miceboth exhibited a large range of fat pad weights, and, on average, fadpad weights increased by approximately 3-fold to 5-fold by the time theanimals reached 9 to 10 months of age. Due to the large range of fat padweights observed in these animals, some animals showed a much largerincrease (up to 10-fold) than others.

In contrast to the wild type and heterozygous knock-out mice, the fatpad weights of myostatin homozygous mutant mice were in a relativelynarrow range and were virtually identical in 2 month old mice and in 9to 10 month old mice. Thus, the increased fat accumulation that occurredwith aging in the wild type mice was not observed in the homozygousmyostatin knock-out mice. This difference in fat accumulation, togetherwith the slight decline in muscle weights, as a function of age in thehomozygous mutant mice fully accounted for the observation that the wildtype animals eventually have the same total body weight as the mutants.

The mean fat pad weights of heterozygous knock-out mice at 9 to 10months of age was intermediate between that of the wild type mice andthe homozygous mutant mice. Although this difference was notstatistically significant, due to the wide range of fat pad weights inthese and the wild type mice, these results nevertheless indicate thatmyostatin has a dose-dependent effect on the accumulation of fat,similar to its effect on muscle growth.

Example 4 Effect of Myostatin on Metabolism

This example demonstrates that serum insulin and glucose levels, as wellas metabolic activity, are affected by the level of myostatinexpression.

In order to determine whether the skeletal muscle hypertrophy and thelack of fat accumulation in the myostatin mutant mice is due to aneffect on overall metabolism, the metabolic profile of the mutant micewas examined. Serum triglyceride and serum cholesterol levels weresignificantly lower in myostatin mutant mice as compared to wild typecontrol mice (Table 1). Serum insulin levels also appeared to be lowerin the myostatin mutant mice. However, the fed and fasting glucoselevels both were indistinguishable among homozygous mutant mice and wildtype mice (Table 1), and both groups of mice had a normal response in aglucose tolerance test. The results demonstrate that the homozygousmyostatin knock-out mice can maintain normal levels of serum glucoseeven though their serum insulin level is lower than that of wild typeanimals.

TABLE 1 SERUM PARAMETERS +/+ −/− triglycerides (mg/dl) 131.5 +/− 16.5 66.8 +/− 11.4 p = 0.012 cholesterol (mg/dl) 138.3 +/− 8.1  94.5 +/− 6.8p = 0.0034 fed glucose (mg/dl) 114.0 +/− 4.8  119.3 +/− 5.2  n.s. (p =0.43) fasting glucose (mg/dl) 86.5 +/− 3.8 103.3 + 9.3 n.s. (p = 0.13)+/+ indicates wild type mice; −/− indicate homozygous knock-out mice

In order to determine whether differences in metabolic rates couldexplain the lack of fat accumulation in the mutant mice, the rate ofoxygen consumption of wild type and mutant mice was compared using acalorimeter. Mutant mice had a lower basal metabolic rate and a loweroverall metabolic rate than wild type control mice. These resultsindicate that the lack of fat accumulation in the myostatin mutant miceis not due to a higher rate of metabolic activity.

Example 5 Myostatin Affects Fat Accumulation in Genetically Obese Mice

This example demonstrates that a lack of myostatin expression suppressesfat accumulation in mice that are a genetic model for obesity.

In order to determine whether the loss of myostatin activity couldsuppress fat accumulation not only in normal mice but also in obesemice, the effect of the myostatin mutation in agouti lethal yellow (Ay)mice, which represent a genetic model of obesity (Yen et al., FASEB J.8:479-488, 1994), was examined. Mice that were doubly heterozygous forthe lethal yellow and myostatin mutations were generated, and offspringfrom crosses of these doubly heterozygous mice were examined.

The total body weight of the Ay/a, myostatin −/− double mutant mouse wasdramatically reduced (approximately 9 grams) compared to that of theAy/a, myostatin +/+ mouse. This reduction in total body weight was evenmore dramatic considering that the Ay/a, myostatin −/− double mutant hadabout 2 to 3 times more skeletal muscle than did the AY/a, myostatin +/+mouse. The double mutant had approximately 10 grams more muscle than theAy/a, myostatin +/+ mouse and, therefore, the total weight reduction inthe rest of the tissues was about 19 grams.

The reduction in total body weight resulted from a reduction in overallfat content. As shown in Table 2, the weights of the parametrial andretroperitoneal fat pads were reduced 5-fold to 6-fold in the Ay/a,myostatin −/− double mutant as compared to the Ay/a, myostatin +/+mouse. These results indicate that the presence of the myostatinmutation dramatically suppresses fat accumulation in obesity.

The presence of the myostatin mutation also dramatically affectedglucose metabolism. Agouti lethal mice lacking the myostatin mutationhad grossly abnormal glucose tolerance test results, with serum glucoselevels often reaching 450 to 600 mg/dl and only slowly recovering tobaseline levels over a period of 4 hours. Female agouti lethal mice wereaffected less than male mice, and some females responded almost normallyin this test, as previously described (see Yen et al., supra, 1994). Incontrast, although the Ay/a, myostatin −/− mice had slightly abnormalglucose tolerance tests, but none of these animals had the grossabnormalities observed in the Ay/a myostatin +/+ mice.

These results indicate that the myostatin mutation at least partiallysuppressed the development of abnormal glucose metabolism in the agoutilethal mice. Significantly, mice that were heterozygous for themyostatin mutation had an intermediate response compared to myostatin+/+ and myostatin −/− mice, thus confirming the dose-dependent effect ofmyostatin.

Example 6 Purification of Recombinant Myostatin

This example provides a method for preparing and isolating recombinantmyostatin.

In order to elucidate the biological activity of myostatin, largequantities of myostatin protein were purified for bioassays. StableChinese hamster ovary (CHO) cell lines producing high levels ofmyostatin protein were generated by co-amplifying a myostatin expressioncassette with a dihydrofolate reductase cassette using a methotrexateselection scheme (McPherron et al., supra, 1997). Myostatin was purifiedfrom the conditioned medium of the highest producing line by successivefractionation on hydroxyapatite, lentil lectin SEPHAROSE, DEAE agarose,and heparin SEPHAROSE. Silver stain analysis revealed that the purifiedprotein obtained following these four column chromatography steps(referred to as “heparin eluate”) consisted of two species withmolecular masses of approximately 35 kilodaltons (kDa) and 12 kDa.

The purified protein preparation was determined by various criteria torepresent a complex of two myostatin prodomain peptides and adisulfide-linked dimer of mature C-terminal myostatin peptides. First,western blot analysis, using antibodies raised against specific portionsof the promyostatin sequence, identified the 35 kDa band as theprodomain and the 12 kDa band as the mature C-terminal peptide. Second,under non-reducing conditions, the species reacting with antibodiesdirected against the mature C-terminal peptide had an electrophoreticmobility consistent with a disulfide linked dimer. Third, the molarratio of prodomain to mature C-terminal peptide was approximately 1:1.Fourth, the prodomain and mature C-terminal peptide copurified throughthe four column chromatography steps. Finally, the mature C-terminalpeptide bound to the lentil lectin column even though the C-terminalregion does not contain consensus N-linked glycosylation signals,indicating that the mature C-terminal peptide bound to the column due toits interaction with the prodomain peptide, which contains a potentialN-linked glycosylation site.

These results indicate that myostatin produced by the geneticallymodified CHO cells is secreted in a proteolytically processed form, andthat the resulting prodomain and mature C-terminal region associatenon-covalently to form a complex containing two prodomain peptides and adisulfide-linked dimer of C-terminal proteolytic fragments, similar tothat described for TGF-β. In the TGF-θ complex, the C-terminal dimerexists in an inactive, latent form (Miyazono et al., J. Biol. Chem.263:6407-6415, 1988), and the active species can be released from thislatent complex by treatment with acid, chaotropic agents, reactiveoxygen species, or plasmin, or by interactions with other proteins,including thrombospondin and integrin Ivβ6 (Lawrence et al., Biochem.Biophys. Res. Comm. 133:1026-1034, 1985; Lyons et al., J. Cell Biol.106:1659-1665, 1988; Schultz-Chemy and Murphy-Ullrich, J. Cell Biol.122:923-932, 1993; Barcellos-Hoff and Dix, Mol. Endocrinol.10:1077-1083, 1996; Munger et al., Cell 96:319-328, 1999). Furthermore,the addition of purified prodomain peptide (also known aslatency-associated peptide or LAP) to the TGF-θ complex inhibits thebiological activity of the purified C-terminal dimer in vitro and invivo (Gentry and Nash, Biochemistry 29:6851-6857, 1990; Bottinger etal., Proc. Natl. Acad. Sci., USA 93:5877-5882, 1996).

The heparin eluate, which consisted of a complex of prodomain and matureC-terminal peptide, was further purified using an HPLC C4 reversed phasecolumn. The C-terminal dimer eluted from the HPLC column earlier thanthe prodomain, thus allowing the isolation of the C-terminal dimer freeof prodomain. Fractions that contained mostly prodomain also wereobtained, although these fractions contained small amounts of theC-terminal dimer. Some of the protein also was present as highermolecular weight complexes. The nature of the higher molecular weightcomplexes is unknown, but, based on western blot analysis in thepresence or absence of reducing agents, these complexes can contain atleast one prodomain peptide and one C terminal mature myostatin peptidelinked by one or more disulfide bonds. In fact, most of the matureC-terminal peptide present in the HPLC fraction enriched for thepropeptide (HPLC fractions 35-37) was present in these high molecularweight complexes. These higher molecular weight complexes likelyrepresent improperly folded proteins that are secreted by thegenetically modified CHO cells.

Example 7 Myostatin Specifically Interacts with an Activin Receptor

This example demonstrates that myostatin specifically binds an activintype II receptor expressed on cells in culture, and that this specificbinding is inhibited by a myostatin prodomain.

The receptors for some TGF-β family members have been identified, andmost are single membrane spanning serine/threonine kinases (Massague andWeis-Garcia, Cancer Surveys 27:41-64, 1996). The activin type IIreceptors (Act RIIA and/or Act RIIB), for example, are known to bindmembers of the TGF-β superfamily. The phenotype of mice lacking the ActRIIB receptor showed anterior/posterior axial patterning defects andkidney abnormalities that were very similar to those observed in GDF-11knock-out mice (McPherron et al., Nat. Genet. 22:260-264, 1999; Oh andLi, Genes Devel. 11:1812-1826, 1997). Since the amino acid sequences ofGDF-11 and myostatin (GDF-8) are 90% identical in the mature C-terminalregion, the ability of myostatin to specifically interact with a activintype II receptor was examined.

Myostatin was labeled by radio-iodination, and binding studies wereperformed using COS cells transfected with an Act RIIB expressionconstruct. Myostatin interacted specifically with the transfected COScells. Myostatin binding was competed in a dose dependent manner byexcess unlabeled myostatin, and was significantly lower in control COScells, which were transfected with an empty vector. No significantbinding occurred to cells transfected with a BMP RII or TGF-β RIIexpression construct. Myostatin binding to Act RIIB-transfected cellswas saturable, and the binding affinity was approximately 5 nM asdetermined by Scatchard analysis.

The receptor binding assay also was used to examine the ability of themyostatin prodomain to inhibit the ability of the mature C-terminaldimer to interact specifically with Act RIIB in this system. Theaddition of purified prodomain peptide blocked the ability of theC-terminal dimer to bind the Act RIIB transfected COS cells in adose-dependent manner. These results indicate that the myostatinprodomain is a natural inhibitor of myostatin.

Example 8 Increased Myostatin Levels Induce Weight Loss

This example demonstrates that elevated levels of myostatin can lead tosubstantial weight loss in vivo.

In one set of experiments, CHO cells that express myostatin wereinjected into nude mice. The nude mice that had myostatin expressing CHOcell tumors showed severe wasting over the course of approximately 12 to16 days following injection of the cells. This wasting syndrome was notobserved in nude mice injected with any of a variety of control CHOlines that had undergone a similar selection process but did not expressmyostatin. Furthermore, the myostatin coding sequence in the constructused to transfect the CHO cells was under the control of ametallothionein promoter, and the wasting syndrome was exacerbated whenmice bearing the myostatin expressing tumors were maintained on watercontaining zinc sulfate. Western blot analysis revealed high levels ofmyostatin protein in the serum of the nude mice that bore themyostatin-expressing CHO cells. These results indicate that the wastingsyndrome was induced in response to the elevated level of myostatin inthe nude mice and, as discussed below, this result was confirmed byobserving similar effects in mice injected with purified myostatin.

The dramatic weight loss observed in the nude mice bearing myostatinexpressing CHO cells was due primarily to a disproportionate loss ofboth fat and muscle weight. White fat pad weights (intrascapular white,uterine, and retroperitoneal fat) were reduced by greater than 90%compared to mice bearing control CHO cell tumors. Muscle weights werealso severely reduced, with individual muscles weighing approximatelyhalf as much in myostatin expressing mice as in control mice by day 16.This loss in muscle weight was reflected by a corresponding decrease infiber sizes and protein content.

Mice bearing the myostatin expressing CHO cell tumors also becameseverely hypoglycemic. However, the weight loss and hypoglycemia werenot due to a difference in food consumption, as all of the mice consumedequivalent amounts of food at each time interval examined during the 16day course of the study. These results indicate that myostatinoverexpression induces a dramatic weight loss, which resembles thecachectic wasting syndrome that occurs in patients suffering fromchronic diseases such as cancer or AIDS.

With more chronic administrations using lower doses of myostatin,changes in fat weight were observed. For example; twice daily injectionsof 1 μg of myostatin protein for 7 days resulted in an approximately 50%decrease in the weights of a number of different white fat pads(intrascapular white, uterine, and retroperitoneal fat pads) with nosignificant effect on brown fat (intrascapular brown). These resultsconfirm that myostatin can induce weight loss and, in extreme cases, awasting syndrome in vivo.

Example 9 Characterization of Myostatin Binding to an Activin Receptor

This example describes a method for characterizing the relationship ofmyostatin binding to an activin receptor with the biological effectsproduced by myostatin in vivo.

Act RIIA or Act RIIB knock-out mice can be used to confirm that Act RIIAor Act RIIB is a receptor for myostatin in vivo. A detailed muscleanalysis of these mice can determine whether knock-out of an activinreceptors is associated with a change in muscle fiber number or size.Since Act RBA/Act RIIB double homozygous mutant die early duringembryogenesis (Song et al., Devel. Biol. 213:157-169, 1999), only thevarious homozygous/heterozygous combinations can be examined. However,tissue-specific or conditional knock-out mice can be generated such thatboth genes can be “deleted” only in muscle tissue, thus allowingpostnatal examination of the double homozygous knock-out mice.

The effect on adipose tissue can be examined with aging of the mice todetermine whether the number of adipocytes or the accumulation of lipidby these adipocytes is altered in the knock-out mice. Adipocyte numberand size is determined by preparing cell suspensions from collagenasetreated tissue (Rodbell, J. Biol. Chem. 239:375-380, 1964; Hirsch andGallian, J. Lipid Res. 9:110-119, 1968). Total lipid content in theanimals is determined by measuring dry carcass weights and then theresidual dry carcass weights after lipid extraction (Folch et al., J.Biol. Chem. 226:497-509, 1957).

A variety of serum parameters also can be examined, including fed andfasting glucose and insulin, triglycerides, cholesterol, and leptin. Asdisclosed above, serum triglycerides and serum insulin are decreased inthe myostatin mutant animals. The ability of the activin receptorknock-out mice to respond to an exogenous glucose load also can beexamined using glucose tolerance tests. As disclosed above, the responseto a glucose load essentially was identical in wild type and myostatinmutant mice at 5 months of age. This observation can be extended bymeasuring these parameters in the mice as they age. Serum insulin levelsalso can be measured at various times during the glucose tolerancetests.

Basal metabolic rates also can be monitored using a calorimeter(Columbus Instruments). As disclosed above, myostatin mutant mice have alower metabolic rate at 3 months of age than their wild typecounterparts. This analysis can be extended to older mice, and therespiratory quotient also can be measured in these animals. The abilityto maintain normal thermogenesis can be determined by measuring thebasal body temperatures as well as their ability to maintain bodytemperature when placed at 4° C. Brown fat weights and expression levelsof UCP1, UCP2, and UCP3 in brown fat, white fat, muscle, and othertissues also can be examined (Schrauwen et al., 1999).

Food intake relative to weight gain can be monitored, and feedefficiency can be calculated. In addition, the weight gain of theanimals placed on high fat diets can be monitored. Wild type micemaintained on a high fat diet accumulate fat rapidly, whereas theresults disclosed herein indicate the activin receptor mutant animalswill remain relatively lean.

The results of these studies can provide a more complete profile of theeffect of myostatin mice, particularly with respect to their overallmetabolic state, thereby providing insight as to whether the ability ofthe myostatin knock-out mice to suppress the accumulation of fat is ananabolic effect of the myostatin mutation in muscle that leads to ashift in energy utilization such that little energy is available forstorage in the form of fat. For example, the decreased fat accumulationcan be due to an increased rate of thermogenesis. These results alsowill provide a baseline for comparison of the effect of the myostatinactivity in the context of different genetic models of obesity and typeII diabetes.

Example 10 Characterization of Myostatin Effects in Genetic Models ofObesity and Type II Diabetes

This example describes methods for determining the effect of myostatinin treating obesity or type II diabetes.

The dramatic reduction in overall fat accumulation in myostatin mutantmice as compared to wild type mice indicates that myostatin activity canbe manipulated to treat or prevent obesity or type II diabetes. Theeffect of the myostatin mutation can be examined in the context ofseveral well characterized mouse models of these metabolic diseases,including, for example, “obese” mice (ob/ob), “diabetic” mice (db/db),and agouti lethal yellow (Ay) mutant strains. Each of these strains isabnormal for virtually every parameter and test described above (see,for example, Yen et al., supra, 1994, Friedman and Halaas, Nature395:763-770, 1998). The ability of the myostatin mutation to slow orsuppress the development of these abnormalities in mice carrying theseother mutations can be examined by constructing double mutants, thensubjecting the double mutant animals, along with appropriate controllittermates carrying only the ob/ob, db/db, or agouti lethal yellowmutations, to the various tests.

As disclosed above, the myostatin mutation in Ay mice was associatedwith about a 5-fold suppression of fat accumulation in the myostatinmutant Ay mice, and with a partial suppression of the development ofabnormal glucose metabolism as assessed by glucose tolerance tests.These results can be extended to include additional animals at variousages, and similar studies can be performed with the ob/ob and db/dbmutants. As both the these mutations are recessive, mice that are doublyhomozygous for the myostatin mutation and either the ob or db mutationcan be generated. In order to examine the effects of partial loss ofmyostatin function in these genetic model systems, mice that arehomozygous for the ob or db mutation and heterozygous for the myostatinmutation also are examined. Mice that are doubly heterozygous for themyostatin and ob mutations have been generated, and the offspring frommatings of these doubly heterozygous mice can be examined, particularlywith respect to fat accumulation and glucose metabolism. Partialsuppression of either or both of these abnormalities in the obesemutants can indicate that myostatin is a target for the treatment ofobesity and type II diabetes.

Example 11 Characterization of Transgenic Mice Expressing DominantNegative Polypeptides that can Affect Myostatin Activity

This example describes methods for characterizing the effect ofmyostatin postnatally by expressing dominant negative polypeptides thatcan block myostatin expression or myostatin signal transduction.

Myostatin Inhibitors

The modulation of myostatin activity postnatally can be used todetermine the effect of myostatin on muscle fiber number (hyperplasia)and muscle fiber size (hypertrophy). Conditional myostatin knock-outmice, in which the myostatin gene is deleted at defined times during thelife of the animal, can be used for these studies. The tet regulator incombination with the cre recombinase provides a system for generatingsuch mice. In this system, the expression of cre is induced byadministration of doxycycline.

Transgenic mice expressing an inhibitor of myostatin from an induciblepromoter also can be generated such that myostatin activity can bereduced or inhibited at defined times during the life of the animal. Thetetracycline regulators are useful for generating such transgenic mice,in which the myostatin expression is induced by doxycycline.

A modification of the tet system, which utilizes co-expression of ahybrid reverse tet-transactivator (fusion protein of the activationdomain of VP16 with the mutant reverse tet repressor) and a hybridtet-transrepressor (fusion protein of the KRAB repressor domain ofmammalian Koxl with the native tet repressor), can be particularlyuseful for producing the transgenic mice (Rossi et al., Nat. Genet.20:389-393, 1998; Forster et al., Nucl. Acids Res. 27:708-710, 1999). Inthis system, the hybrid reverse tet-transactivator binds tet operatorsequences, and activates transcription only in the presence oftetracycline; the hybrid tet-transrepressor binds tet operator sequencesand represses transcription only in the absence of tetracycline. Byco-expressing these two fusion proteins, the basal activity of thetarget promoter is silenced by the tet-transrepressor in the absence oftetracycline, and is activated by the reverse tet-transactivator uponadministration of tetracycline.

Two types of transgenic lines can be generated. In the first type, thetransgene encodes a myostatin inhibitor polypeptide under the control ofa muscle specific promoter, for example, the muscle creatine kinasepromoter (Sternberg et al., supra, 1988) or the myosin light chainenhancer/promoter (Donoghue et al., supra, 1991). Individual transgeniclines are screened for specific expression of the tet regulators inskeletal muscle, and several independent lines for each of the twopromoters are selected and examined to confirm that any effects observedare not due, for example, to integration site-specific effects. Aconstruct containing the two tet regulators under the control of themyosin light chain promoter/enhancer has been constructed, and can beused for pronuclear injections. In the second type of line. thetransgene contains a myostatin inhibitor polypeptide under the controlof a minimal CMV promoter that further contains tet operator sequences.

The myostatin inhibitor can be a dominant negative form of myostatin ora myostatin prodomain, which, as disclosed herein, can inhibit myostatinactivity. Dominant negative forms of TGF-β family members have beendescribed (see, for example, Lopez et al., Mol. Cell. Biol.12:1674-1679, 1992; Wittbrodt and Rosa, Genes Devel. 8:1448-1462, 1994),and contain, for example, a mutant proteolytic cleavage site, therebypreventing the protein from being processed into the biologically activespecies. When co-expressed in a cell with the endogenous wild type gene,the mutant protein forms non-functional heterodimers with the wild typeprotein, thus acting as a dominant negative. A mutant myostatinpolypeptide containing a mutation in the promyostatin cleavage site hasbeen constructed, and can be examined for a dominant negative effect byco-expressing the mutant with wild type myostatin in varying ratios in293 cells. Conditioned medium from 293 cells transiently transfectedwith the constructs can be examined by western blot analysis and theability of the mutant to block the formation of mature C-terminal dimerscan be examined.

An expression construct encoding only the myostatin prodomain also canbe utilized. As disclosed above, the prodomain forms a tight complexwith the mature C-terminal dimer and blocks the ability of the matureC-terminal myostatin dimer to bind Act RIIB in cells expressing thereceptor in culture. By analogy to TGF-β, the myostatin prodomain alsocan maintain the mature C-terminal dimer in an inactive latent complexin vivo.

These transgenic animals can be bred with those expressing the tetregulators to generate doubly transgenic lines containing both the tetregulators and the inhibitor target construct. These doubly transgeniclines can be screened for those in which all of the different componentsare expressed appropriately. Northern blot analysis using RNA obtainedfrom various muscles and control tissues from representative mice ineach line, before and after administration of doxycycline in thedrinking water, can be used to identify such transgenic lines.Transgenic lines will be selected that do not express the transgene inany tissue in the absence of doxycycline, and that express the transgeneonly in muscle in the presence of doxycycline.

Doxycycline is administered to the selected transgenic animals and theeffect on muscle mass is examined. Doxycycline can be administered topregnant mothers to induce the expression of the inhibitor duringembryogenesis. The effect of blocking myostatin activity duringdevelopment of the transgenic animals can be compared to the effectsobserved in myostatin knock-out mice. Since the promoters for drivingexpression of the tet regulators can be induced at a later time duringdevelopment than the time when myostatin is first expressed, the effecton muscle mass in the transgenic mice can be compared to the effect thatoccurs in the myostatin knock-out mice.

The effect of inhibiting myostatin activity postnatally can be examinedby administering doxycycline to the doubly transgenic mice at varioustimes after birth. Doxycycline treatment can begin, for example, at 3weeks of age, and the animals can be analyzed at 5 months of age, whichis the age at which the difference in muscle weights were at a maximumin the myostatin knock-out mice versus wild type mice. The animals areexamined for the effects of the inhibitor on muscle mass. Muscles alsocan be examined histologically to determine effects on fiber number andfiber size. In addition, a fiber type analysis of various muscles in thetransgenic mice can be performed to determine whether there is aselective effect on type I or type II fibers.

Doxycycline can be administered in different doses and at differenttimes to characterize the effect of the myostatin inhibitors. Doublytransgenic mice also can be maintained chronically on doxycycline, thenexamined for effects on fat pad weights and other relevant metabolicparameters as described above. The results of these studies can confirmthat modulating myostatin activity postnatally can increase muscle massor decrease fat accumulation, thus indicating that targeting myostatincan be useful for the treatment of a variety of muscle wasting andmetabolic diseases clinically.

Myostatin

Transgenic mice containing a myostatin transgene also can be examinedand the effects produced upon expression of myostatin can be comparedwith those observed in the nude mice containing the myostatin-expressingCHO cells. Similarly as described above, myostatin can be placed undercontrol of conditional (tet) and tissue specific regulatory elements,and expression of myostatin in the transgenic mice can be examined todetermine whether a wasting syndrome occurs similar to that observed inthe nude mice. The myostatin transgene can include, for example,processing signals derived from SV40, such that transgene can bedistinguished from the endogenous myostatin gene.

Serum samples can be isolated from the myostatin transgenic mice atvarious times following the administration of doxycycline and the levelof myostatin transgene product in the serum can be determined. Totalbody weights of the animals are monitored over time to determine whetherthe animals show significant weight loss. In addition, individualmuscles and fat pads are isolated and weighed, and the number, size andtype of muscle fibers are determined in selected muscle samples.

The level of myostatin transgene expression can be varied by varying thedose of doxycycline administered to the animals. Transgene expressioncan be monitored using, for example, northern blot analysis of transgeneRNA levels in muscle, or myostatin protein levels in serum. Theidentification of specific levels of myostatin transgene expressionallows a correlation of the extent of wasting induced by myostatin. Thetransgenic lines also can be crossed with the myostatin knock-out miceto generate mice in which the only source of myostatin is expressionfrom the transgene. Expression of myostatin at various times duringdevelopment can be examined and the effect of myostatin on fiber number,fiber size, and fiber type can be determined. The availability of micein which the expression of myostatin can be precisely and rapidlycontrolled provides a powerful tool for further characterizing themyostatin signal transduction pathway and for examining the effects ofvarious agents that potentially can be useful for modulating myostatinsignal transduction.

Effectors of Myostatin Signal Transduction

Transgenic mice containing either dominant negative forms of a myostatinsignal transduction pathway, which can include components of a TGF-θsignal transduction pathway, that is expressed specifically in skeletalmuscle can be generated. As disclosed herein, the Smad proteins, whichmediate signal transduction through a pathway induced by activin type IIreceptors, can be involved in myostatin signal transduction.

Act RIIB can bind GDF-11, which is highly related to myostatin(McPherron et al., supra, 1997; Gamer et al., supra, 1999; Nakashima etal., Mech. Devel. 80:185-189, 1999), and expression of c-ski, which canbind an inhibit Smad 2, Smad 3, and Smad 4, dramatically affects musclegrowth (Suprave et al., supra, 1990; Berk et al., supra, 1997; see,also, Luo et al., supra, 1999; Stroschein et al., supra, 1999; Sun etal., supra, 1999a and b; Akiyoshi et al., supra, 1999). As disclosedherein, myostatin interacts specifically with Act RIIB and, therefore,can exert its biological effect, at least in part, by binding to activintype II receptors in vivo and activating the Smad signaling pathway.

The role of the Smad signaling pathway in regulating muscle growth canbe examined using transgenic mouse lines that are blocked, or capable ofbeing blocked, at specific points in the Act RIIB/Smad signaltransduction pathway. The muscle creatine kinase promoter or myosinlight chain enhancer/promoter can be used to drive expression of variousinhibitors of the Smad signal transduction pathway.

An inhibitor useful in this system can include, for example,follistatin; a dominant negative Act RIIB receptor; a dominant negativeSmad polypeptide such as Smad 3; c-ski; or an inhibitory Smadpolypeptide such as Smad 7. Follistatin can bind and inhibit theactivity of certain TGF-β family members, including GDF-11 (Gamer etal., supra, 1999). Dominant negative forms of an activin type IIreceptor can be obtained by expressing a truncated GDF receptor, forexample, by expressing the extracellular domain, particularly a solubleform of an Act RIIB extracellular domain, or by expressing a truncatedAct RIIB receptor that lacks the kinase domain or contains a mutationsuch that the mutant receptor lacks kinase activity. Smad 7 functions asan inhibitory Smad that can block the signaling pathway induced byactivin, TGF-β, and BMP. A dominant negative form of Smad 3, forexample, can be constructed by mutating the Smad 3 C-terminalphosphorylation sites, thereby blocking Smad 3 function (Liu et al.,supra, 1997). c-ski overexpression has been correlated to musclehypertrophy in transgenic mice (Suprave et al., supra, 1990).

Transgenic mice can be prepared and each founder line examined forproper, muscle-specific expression of the transgene. The selected miceare examined for total body weights, individual muscle weights, andmuscle fiber sizes, numbers and types. Those lines demonstrating a cleareffect on muscle mass can be examined further for fat accumulation andother relevant metabolic parameters as described above. The use of thesedifferent agents to target specific steps in the activin receptor/Smadsignal transduction pathway is particularly informative because thesignaling pathways for the different agents overlap at different steps.For example, follistatin binds to and inhibits activin and GDF-11activity, but not TGF-β, whereas a dominant negative Smad 3 can blocksignaling through both activin and TGF-β receptors. Smad 7 can have aneven more pleotropic because it blocks signaling through BMP receptorsas well. The studies can allow the identification of specific targetsfor modulating myostatin activity, thus providing various strategies fordeveloping drugs or other agents that modulate myostatin signaltransduction and, therefore, myostatin activity.

In particular, the transgenic lines described herein can be used todetermine the effect of blocking myostatin function or the Smadsignaling pathway postnatally on the development of obesity or type IIdiabetes. For example, the inhibitory transgenes can be crossed into theob/ob, db/db, and Ay mutant mice. In the absence of doxycycline, aninhibitor transgene is not expressed and, therefore, the animals areindistinguishable from each of the parental mutant mice. In the presenceof doxycycline, the inhibitor is expressed and can block myostatinactivity. The effect of blocking myostatin activity on development ofthe metabolic abnormalities in these mutant animals can be examined.

Expression of the inhibitor can be induced at an early age, for example,at 3 weeks of age, to maximize the effect. In addition, myostatinactivity can be blocked prior to the time that the metabolicabnormalities become so severe as to be irreversible. Animals can bemaintained on doxycycline and assessed at various ages using the testsdescribed above, including those relating to fat accumulation andglucose metabolism. Any delay in the age at which one or more testresults becomes abnormal in the ob/ob, db/db, and Ay mutant animals canbe identified. Similar studies can be performed using older animals,which have developed some of the signs of obesity or type II diabetes,and the effect of blocking myostatin activity on various parameters,including fat weight and glucose metabolism, can be determined. Theresults of these studies can further identify specific targets that canbe manipulated in an effort to prevent or treat obesity or type IIdiabetes.

Example 12 Characterization of Myostatin Effect on the Induction ofCachexia

This example describes methods for determining the role of myostatinsignal transduction in the development and progression of cachexia.

The activin receptor and Smad pathway can constitute at least part ofthe signal transduction pathway involved in mediating myostatin activityin normal individuals and, therefore, can be involved in mediating theeffects that occur in an individual due to excess levels of myostatin.As disclosed herein, cachexia, for example, can be mediated, at least inpart, by abnormally high levels of myostatin. As such, methods formanipulating signal transduction through the Smad pathway can provide anew strategy for developing drugs for the treatment of muscle wasting ingeneral and cachexia in particular.

The role of the Smad signaling pathway in cachexia can be examined byexamining the susceptibility of the various transgenic lines describedabove to cachexia, which can be induced, for example, by interleukin-6(IL-6; Black et al., Endocrinology 128:2657-2659, 1991, which isincorporated herein by reference]), tumor necrosis factor-I (TNF-I;Oliff et al., Cell 50:555-563, 1987, which is incorporated herein byreference), or certain tumor cells. In the case of IL-6 and TNF-I, theinhibitor transgenes can be crossed into a nude mouse background, thenthe animals can be challenged with CHO cells that produce IL-6 or TNF-I,which induce wasting in nude mice when overexpressed in this manner. CHOcells that overproduce IL-6 or TNF-I can be prepared using the methodsdescribed above for generating myostatin overproducing cells. Forexample, TNF-I cDNA can be cloned into the pMSXND expression vector (Leeand Nathans, J. Biol. Chem. 263:3521-3527, 1988), then cells carryingamplified copies of the expression construct can be selected stepwise inincreasing concentrations of methotrexate.

Tumor cells such as Lewis lung carcinoma cells (Matthys et al., Eur. J.Cancer 27:182-187, 1991, which is incorporated herein by reference) orcolon 26 adenocarcinoma cells (Tanaka et al., J. Cancer Res.50:2290-2295, 1990, which is incorporated herein by reference), whichcan induce cachexia in mice, also can be utilized for these studies.These cell lines cause severe wasting when grown as tumors in mice.Thus, the effect of these tumors can be examined in the varioustransgenic mice described herein. It is recognized that the varioustumor cells will only grow in certain genetic backgrounds. For example,the Lewis lung carcinoma cells are routinely grown in C57 BL/6 mice, andthe colon 26 carcinoma cells are routinely grown in BALB/c mice. Thus,the transgenes can be backcrossed into these or other geneticbackgrounds to allow growth of the tumor cells.

Various parameters, including total body weight, individual muscleweight, muscle fiber size and number, food intake and serum parameters,including glucose levels, can be monitored. In addition, serum myostatinlevels and myostatin RNA levels in muscle can be examined to confirmthat increased myostatin expression is correlated with cachexia. Theresults of these studies can confirm that the action of myostatin isdownstream of the cachexia-inducing agents in these experimental models.The results also can confirm that the Smad signaling pathway isessential for development of cachexia in these models, and candemonstrate that a therapeutic benefit can be obtained in the treatmentof cachexia by modulating the Smad signaling.

Example 13 Identification and Characterization of Growth DifferentiationFactor-8 (GDF-8) and GDF-11 Receptors

This example describes methods for identifying and characterizing cellsurface receptors for GDF-8 (myostatin) and GDF-11.

The purified GDF-8 and GDF-11 proteins will be used primarily to assayfor biological activities. In order to identify potential target cellsfor GDF-8 and GDF-11 action cells expressing their receptors will besearched. For this purpose, the purified protein will be radio-iodinatedusing the chloramine T method, which has been used successfully to labelother members of this superfamily, like TGF-β (Cheifetz et al., supra,1987), activins (Sugino et al., J. Biol. Chem. 263:15249-15252, 1988),and BMPs (Paralkar et al., Proc. Natl. Acad. Sci., USA 88:3397-3401,1991), for receptor binding studies. The mature processed forms of GDF-8and GDF-11 each contain multiple tyrosine residues. Two differentapproaches will be taken to identify receptors for these proteins.

One approach will determine the number, affinity, and distribution ofreceptors. Either whole cells grown in culture, frozen sections ofembryos or adult tissues, or total membrane fractions prepared fromtissues or cultured cells will be incubated with the labeled protein,and the amount or distribution of bound protein will be determined. Forexperiments involving cell lines or membranes, the amount of bindingwill be determined by measuring either the amount of radioactivity boundto cells on the dish after several washes or, in the case of membranes,the amount of radioactivity sedimented with the membranes aftercentrifugation or retained with the membranes on a filter. Forexperiments involving primary cultures, where the number of cells can bemore limited, binding sites will be visualized directly by overlayingwith photographic emulsion. For experiments involving frozen sections,sites of ligand binding will be visualized by exposing these sections tohigh resolution Beta-max hyperfilm; if finer localization is required,the sections will be dipped in photographic emulsion. For all of theseexperiments, specific binding will be determined by adding excessunlabeled protein as competitor (for example, see Lee and Nathans,supra, 1988).

A second approach will be to characterize the receptor biochemically.Membrane preparations or potential target cells grown in culture will beincubated with labeled ligand, and receptor/ligand complexes will becovalently cross-linked using disuccinimidyl suberate, which has beencommonly used to identify receptors for a variety of ligands, includingmembers of the TGF-β superfamily (Massague and Like, J. Biol. Chem.260:2636-2645, 1985). Cross-linked complexes are separated byelectrophoresis on SDS polyacrylamide gels to look for bands labeled inthe absence, but not in the presence, of excess unlabeled protein. Themolecular weight of the putative receptor will be estimated bysubtracting the molecular weight of the ligand. An important questionthat these experiments will address is whether GDF-8 and GDF-11 signalthrough type I and type II receptors like many other members of theTGF-β superfamily (Massague and Weis-Garcia, supra, 1996).

Once a method for detecting receptors for these molecules has beenachieved, more detailed analysis will be carried out to determine thebinding affinities and specificities. A Scatchard analysis will be usedto determine the number of binding sites and dissociation constants. Bycarrying out cross-competition analyses between GDF-8 and GDF-11, itwill be possible to determine whether they are capable of binding to thesame receptor and their relative affinities. These studies will give anindication as to whether the molecules signal through the same ordifferent receptors. Competition experiments using other TGF-β familymembers will be performed to determine specificity. Some of theseligands are available commercially, and some others are available fromGenetics Institute, Inc.

For these experiments, a variety of embryonic and adult tissues and celllines will be tested. Based on the specific expression of GDF-8 inskeletal muscle and the phenotype of GDF-8 knock-out mice, initialstudies focus on embryonic and adult muscle tissue for membranepreparation and for receptor studies using frozen sections. In addition,myoblasts will be isolated and cultured from embryos at various days ofgestation or satellite cells from adult muscle as described (Vivarelliand Cossu, Devel. Biol. 117:319-325, 1986; Cossu et al., Cell Diff.9:357-368, 1980). The binding studies on these primary cells aftervarious days in culture will be performed and binding sites localized byautoradiography so that the binding sites can be c-o-localized withvarious myogenic markers, such as muscle myosin (Vivarelli et al., J.Cell Biol. 107:2191-2197, 1988), and correlate binding with thedifferentiation state of the cells, such as formation of multinucleatedmyotubes. In addition to using primary cells, cell lines will beutilized to look for receptors. In particular, the initial focus will beon three cells lines, C2C12, L6, and P19. C2C12 and L6 myoblastsdifferentiate spontaneously in culture and form myotubes depending onthe particular growth conditions (Yaffe and Saxel, supra, 1977; Yaffe,supra, 1968). P19 embryonal carcinoma cells can be induced todifferentiate into various cell types, including skeletal muscle cellsin the presence of DMSO (Rudnicki and McBurney, Teratocarcinomas andEmbryonic Stem Cells: A practical approach (E. J. Robertson, IRL Press,Cambridge 1987). Receptor binding studies will be carried out on thesecell lines under various growth conditions and at various stages ofdifferentiation. Although the initial studies will focus on musclecells, other tissues and cell types will be examined for the presence ofGDF-8 and GDF-11 receptors.

Recombinant human GDF-8 (rhGDF-8) homodimer will be used in thesebinding studies. RhGDF-8 was expressed using CHO cells and purified toapproximately 90% purity. The rhGDF-8 had the expected 25 kDa to 27 kDamolecular weight and, upon reduction, was reduced to the 12 kDa monomer.Using 1-125 labeled GDF-8 in a receptor-ligand binding assay, twomyoblast cell lines, L6 and G-8, bound GDF-8. The binding was specificsince non labeled GDF-8 effectively competed the binding of the labeledligand. The dissociation constant (Kd) was 370 pM, and L6 myoblasts havea high number (5,000 receptors/cell) of cell surface binding proteins.GDF-11 (BMP-11) is highly homologous (>90%) to GDF-8. Receptor bindingstudies revealed that GDF-8 and GDF-11 bound to the same bindingproteins on L6 myoblasts. It is important to establish whether or notGDF-8 binds to the known TGF-9 receptor. TGF-9 did not compete thebinding of GDF-8, indicating that the GDF-8 receptor is distinct fromthe TGF-9 receptor. The GDF-8 receptor was not expressed on all myoblastcell lines, including four myoblast cell lines, C2C12, G7, MLB13MYC c14and BC3H1, which do not bind GDF-8.

The gene or genes encoding receptors for GDF-8 and GDF-11 can beobtained. As a first step towards understanding the mechanism by whichGDF-8 and GDF-11 exert their biological effects, it is important toclone the genes encoding their receptors. From the experiments above, itwill be more clear as to whether GDF-8 and GDF-11 bind to the samereceptor or to different receptors. There will also be considerableinformation regarding the tissue and cell type distribution of thesereceptors. Using this information, two different approaches will betaken to clone the receptor genes.

The first approach will be to use an expression cloning strategy. Infact, this was the strategy that was originally used by Mathews and Vale(Cell 65:973-982, 1991) and Lin et al. (Cell 68:775-785, 1992) to clonethe first activin and TGF-β receptors. Poly A-selected RNA from thetissue or cell type that expresses the highest relative number of highaffinity binding sites will be obtained, and used to prepare a cDNAlibrary in the mammalian expression vector pcDNA-1, which contains a CMVpromoter and an SV40 origin of replication. The library will be plated,and cells from each plate will be pooled into broth and frozen. Aliquotsfrom each pool will be grown for preparation of DNA. Each individualpool will be transiently transfected into COS cells in chamber slides,and transfected cells will be incubated with iodinated GDF-8 or GDF-11.After washing away the unbound protein, the sites of ligand binding willbe visualized by autoradiography. Once a positive pool is identified,the cells from that pool will be replated at lower density, and theprocess will be repeated. Positive pools will then be plated, andindividual colonies will be picked into grids and re-analyzed asdescribed (Wong et al., Science 228:810-815, 1985).

Initially, using pool sizes of 1500 colonies will be screened. In orderto be certain to identify a positive clone in a mixture of thiscomplexity, a control experiment using TGF-β and a cloned type IIreceptor will be performed. The coding sequence for the TGF-β type IIreceptor will be cloned into the pcDNA-1 vector, and bacteriatransformed with this construct will be mixed with bacteria from ourlibrary at various ratios, including 1:1500. The DNA prepared from thismixture then will be transfected into COS cells, incubated withiodinated TGF-β, and visualized by autoradiography. If positive signalsare observed at a ratio of 1:1500, pools of 1500 clones will bescreened. Otherwise, smaller pool sizes corresponding to ratios at whichthe procedure is sensitive enough to identify a positive signal incontrol experiments will be used.

A second parallel strategy to attempt to clone the GDF-8 and GDF-11receptors also will be used, taking advantage of the fact that mostreceptors for members of the TGF-β superfamily that have been identifiedbelong to the membrane-spanning serine/threonine kinase family (Massagueand Weis-Garcia, supra, 1996). Because the cytoplasmic domains of thesereceptors are related in sequence, degenerate PCR probes will be used toclone members of this receptor family that are expressed in tissues thatcontain binding sites for GDF-8 and GDF-11. In fact, this is theapproach that has been used to identify most of the members of thisreceptor family. The general strategy will be to design degenerateprimers corresponding to conserved regions of the known receptors, touse these primers for PCR on cDNA prepared from the appropriate RNAsamples (most likely from skeletal muscle), to subclone the PCRproducts, and finally to sequence individual subclones. As sequences areidentified, they will be used as hybridization probes to eliminateduplicate clones from further analysis. The receptors that areidentified then will be tested for their ability to bind purified GDF-8and GDF-11. Because this screen will yield only small PCR products,full-length cDNA clones will be obtained for each receptor from cDNAlibraries prepared from the appropriate tissue, inserted into thepcDNA-1 vector, transfected into COS cells, and the transfected cellswill be assayed for their ability to bind iodinated GDF-8 or GDF-11.Ideally, every receptor that is identified in this screen will be testedfor the ability to bind these ligands. However, the number of receptorsthat are identified can be large, and isolating all of the full-lengthcDNAs and testing them can require considerable effort. Almost certainlysome of the receptors that are identified will correspond to knownreceptors, and for these, either obtaining full-length cDNA clones fromother investigators or amplifying the coding sequences by PCR based onthe published sequences should be straightforward. For novel sequences,the tissue distribution will be determined by northern blot analysis andthe highest priority will be directed to those receptors whoseexpression pattern most closely resembles the distribution of GDF-8and/or GDF-11 binding sites as determined above.

In particular, it is known that these receptors fall into two classes,type I and type II, which can be distinguished based on the sequence andwhich are both required for full activity. Certain ligands cannot bindtype I receptors in the absence of type II receptors while others arecapable of binding both receptor types (Massague and Weis-Garcia, supra,1996). The cross-linking experiments outlined above should give someindication as to whether both type I and type II receptors are alsoinvolved in signaling GDF-8 and GDF-11. If so, it will be important toclone both of these receptor subtypes in order to fully understand howGDF-8 and GDF-11 transmit their signals. Because it cannot be predictedas to whether the type I receptor is capable of interacting with GDF-8and GDF-11 in the absence of the type II receptor, type II receptor(s)will be cloned first. Only after at least one type II receptor has beenidentified for these ligands, will an attempt be made to identify thetype I receptors for GDF-8 and GDF-11. The general strategy will be tocotransfect the type II receptor with each of the type I receptors thatare identified in the PCR screen, then assay the transfected cells bycrosslinking. If the type I receptor is part of the receptor complex forGDF-8 or GDF-11, two cross-linked receptor species should be detected inthe transfected cells, one corresponding to the type I receptor and theother corresponding to the type II receptor.

The search for GDF-8 and GDF-11 receptors is further complicated by thefact at least one member of the TGF-β superfamily, namely, GDNF, iscapable of signaling through a completely different type of receptorcomplex involving a GPI-linked component (GDNFR-alpha) and a receptortyrosine kinase (c-ret; Trupp et al., Nature 381:785-789, 1996; Durbecet al., Nature 381:789-793, 1996; Treanor et al., Nature 382:80-83,1996; Jing et al., Cell 85:1113-1124, 1996). Although GDNF is the mostdistantly-related member of the TGF-β superfamily, it is certainlypossible that other TGF-β family members can also signal through ananalogous receptor system. If GDF-8 and GDF-11 do signal through asimilar receptor complex, the expression screening approach should beable to identify at least the GPI-linked component (indeed GDNFR-alphawas identified using an expression screening approach) of this complex.In the case of GDNF, the similar phenotypes of GDNF- and c-ret-deficientmice suggested c-ret as a potential receptor for GDNF.

Example 14 Preparation and Characterization of GDF-11 Knock-Out Mice

The phenotype of GDF-11 knock-out mice in several respects resembles thephenotype of mice carrying a deletion of a receptor for some members ofthe TGF-θ superfamily, including the activin type IIB receptor (ActRIIB). To determine the biological function of GDF-11, the GDF-11 genewas disrupted by homologous targeting in embryonic stem cells.

A murine 129 SvJ genomic library was prepared in lambda FIXII accordingto the instructions provided by Stratagene (La Jolla, Calif.). Thestructure of the GDF-11 gene was deduced from restriction mapping andpartial sequencing of phage clones isolated from the library. Vectorsfor preparing the targeting construct were kindly provided by PhilipSoriano and Kirk Thomas. To ensure that the resulting mice would be nullfor GDF-11 function, the entire mature C-terminal region was deleted andreplaced by a neo cassette. R1 ES cells were transfected with thetargeting construct, selected with gancyclovir (2 TM) and G418 (250Tg/ml), and analyzed by Southern blot analysis.

Homologous targeting of the GDF-11 gene was observed in 8/155gancyclovir/G418 doubly resistant ES cell clones. Following injection ofseveral targeted clones into C57BL/6J blastocysts, chimeras wereobtained from one ES clone that produced heterozygous pups when crossedto both C57BL/6J and 129/SvJ females. Crosses of C57BL/6J/129/SvJ hybridF1 heterozygotes produced 49 wild type (34%), 94 heterozygous (66%), andno homozygous mutant adult offspring. Similarly, there were no adulthomozygous null animals seen in the 129/SvJ background (32 wild type(36%) and 56 heterozygous mutant (64%) animals).

To determine the age at which homozygous mutants were dying, litters ofembryos isolated at various gestational ages from heterozygous femalesthat had been mated to heterozygous males were genotyped. At allembryonic stages examined, homozygous mutant embryos were present atapproximately the predicted frequency of 25%. Among hybrid newborn mice,the different genotypes were also represented at the expected Mendelianratio of 1:2:1 (34+/+(28%), 61+/−(50%), and 28−/− (23%)). Homozygousmutant mice were born alive and were able to breath and nurse. Allhomozygous mutants died, however, within the first 24 hours after birth.The precise cause of death was unknown, but the lethality may have beenrelated to the fact that the kidneys in homozygous mutants were eitherseverely hypoplastic or completely absent.

Homozygous mutant animals were easily recognizable by their severelyshortened or absent tails. To further characterize the tail defects inthese homozygous mutant animals, their skeletons were examined todetermine the degree of disruption of the caudal vertebrae. A comparisonof wild type and mutant skeleton preparations of late stage embryos andnewborn mice, however, revealed differences not only in the caudalregion of the animals but in many other regions as well. In nearly everycase where differences were noted, the abnormalities appeared torepresent homeotic transformations of vertebral segments in whichparticular segments appeared to have a morphology typical of moreanterior segments. These transformations were evident throughout theaxial skeleton extending from the cervical region to the caudal region.Except for the defects seen in the axial skeleton, the rest of theskeleton, such as the cranium and limb bones, appeared normal.

Anterior transformations of the vertebrae in mutant newborn animals weremost readily apparent in the thoracic region, where there was a dramaticincrease in the number of thoracic (T) segments. All wild type miceexamined showed the typical pattern of 13 thoracic vertebrae each withits associated pair of ribs. In contrast, homozygous mutant mice showeda striking increase in the number of thoracic vertebrae. All homozygousmutants examined had 4 to 5 extra pairs of ribs for a total of 17 to 18,although in over ⅓ of these animals, the 18th rib appeared to berudimentary. Hence, segments that would normally correspond to lumbar(L) segments L1 to L4 or L5 appeared to have been transformed intothoracic segments in mutant animals.

Moreover, transformations within the thoracic region in which onethoracic vertebra had a morphology characteristic of another thoracicvertebra were also evident. For example, in wild type mice, the first 7pairs of ribs attach to the sternum, and the remaining 6 are unattachedor free. In homozygous mutants, there was an increase in the number ofboth attached and free pairs of ribs to 10-11 and 7-8, respectively.Therefore, thoracic segments T8, T9, T10, and in some cases even T11,which all have free ribs in wild type animals, were transformed inmutant animals to have a characteristic typical of more anteriorthoracic segments, namely, the presence of ribs attached to the sternum.Consistent with this finding, the transitional spinous process andtransitional articular processes which are normally found on T10 in wildtype animals were instead found on T13 in homozygous mutants. Additionaltransformations within the thoracic region were also noted in certainmutant animals. For example, in wild type mice, the ribs derived from T1normally touch the top of the sternum. However, in 2/23 hybrid and ⅔129/SvJ homozygous mutant mice examined, T2 appeared to have beentransformed to have a morphology resembling that of T1; that is, inthese animals, the ribs derived from T2 extended to touch the top of thesternum. In these cases, the ribs derived from T1 appeared to fuse tothe second pair of ribs. Finally, in 82% of homozygous mutants, the longspinous process normally present on T2 was shifted to the position ofT3. In certain other homozygous mutants, asymmetric fusion of a pair ofvertebrosternal ribs was seen at other thoracic levels.

The anterior transformations were not restricted to the thoracic region.The anterior most transformation that we observed was at the level ofthe 6th cervical vertebra (C6). In wild type mice, C6 is readilyidentifiable by the presence of two anterior tuberculi on the ventralside. In several homozygous mutant mice, although one of these twoanterior tuberculi was present on C6, the other was present at theposition of C7 instead. Hence, in these mice, C7 appeared to have beenpartially transformed to have a morphology resembling that of C6. Oneother homozygous mutant had 2 anterior tuberculi on C7 but retained oneon C6 for a complete C7 to C6 transformation but a partial C6 to C5transformation.

Transformations of the axial skeleton also extended into the lumbarregion. Whereas wild type animals normally have only 6 lumbar vertebrae,homozygous mutants had 8 to 9. At least 6 of the lumbar vertebrae in themutants must have derived from segments that would normally have givenrise to sacral and caudal vertebrae as the data described above suggestthat 4 to 5 lumbar segments were transformed into thoracic segments.Hence, homozygous mutant mice had a total of 33-34 presacral vertebraecompared to 26 presacral vertebrae normally present in wild type mice.The most common presacral vertebral patterns were C7/T18/L8 andC7/T18/L9 for mutant mice compared to C7/T13/L6 for wild type mice. Thepresence of additional presacral vertebrae in mutant animals was obviouseven without detailed examination of the skeletons as the position ofthe hind limbs relative to the forelimbs was displaced posteriorly by 7to 8 segments.

Although the sacral and caudal vertebrae were also affected inhomozygous mutant mice, the exact nature of each transformation was notas readily identifiable. In wild type mice, sacral segments S1 and S2typically have broad transverse processes compared to S3 and S4. In themutants, there did not appear to be an identifiable S1 or S2 vertebra.Instead, mutant animals had several vertebrae that appeared to havemorphology similar to S3. In addition, the transverse processes of all 4sacral vertebrae are normally fused to each other although in newbornsoften only fusions of the first 3 vertebrae are seen. In homozygousmutants, however, the transverse processes of the sacral vertebrae wereusually unfused. In the caudal-most region, all mutant animals also hadseverely malformed vertebrae with extensive fusions of cartilage.Although the severity of the fusions made it difficult to count thetotal number of vertebrae in the caudal region, up to 15 transverseprocesses were counted in several animals. It could not be determinedwhether these represented sacral or caudal vertebrae in the mutantsbecause morphologic criteria for distinguishing S4 from caudal vertebraeeven in wild type newborn animals could not be established. Regardlessof their identities, the total number of vertebrae in this region wassignificantly reduced from the normal number of approximately 30. Hence,although the mutants had significantly more thoracic and lumbervertebrae than wild type mice, the total number of segments was reducedin the mutants due to the truncation of the tails.

Heterozygous mice also showed abnormalities in the axial skeletonalthough the phenotype was much milder than in homozygous mice. The mostobvious abnormality in heterozygous mice was the presence of anadditional thoracic segment with an associated pair of ribs. Thistransformation was present in every heterozygous animal examined, and inevery case, the additional pair of ribs was attached to the sternum.Hence, T8, whose associated rib normally does not touch the sternum,appeared to have been transformed to a morphology characteristic of amore anterior thoracic vertebra, and L1 appeared to have beentransformed to a morphology characteristic of a posterior thoracicvertebra. Other abnormalities indicative of anterior transformationswere also seen to varying degrees in heterozygous mice. These included ashift of the long spinous process characteristic of T2 by one segment toT3, a shift of the articular and spinous processes from T10 to T11, ashift of the anterior tuberculus on C6 to C7, and transformation of T2to T1 where the rib associated with T2 touched the top of the sternum.

In order to understand the basis for the abnormalities in axialpatterning seen in GDF-11 mutant mice, mutant embryos isolated atvarious stages of development were examined, and compared o wild typeembryos. By gross morphological examination, homozygous mutant embryosisolated up to day 9.5 of gestation were not readily distinguishablefrom corresponding wild type embryos. In particular, the number ofsomites present at any given developmental age was identical betweenmutant and wild type embryos, suggesting that the rate of somiteformation was unaltered in the mutants. By day 10.5-11.5 p.c., mutantembryos could be easily distinguished from wild type embryos by theposterior displacement of the hind limb by 7-8 somites. Theabnormalities in tail development were also readily apparent at thisstage. Taken together, these data suggest that the abnormalitiesobserved in the mutant skeletons represented true transformations ofsegment identities rather than the insertion of additional segments, forexample, by an enhanced rate of somitogenesis.

Alterations in expression of homeobox containing genes are known tocause transformations in Drosophila and in vertebrates. To see if theexpression patterns of Hox genes (the vertebrate homeobox containinggenes) were altered in GDF-11 null mutants, the expression pattern of 3representative Hox genes, Hoxc-6, Hoxc-8 and Hoxc-11, was determined inday 12.5 p.c. wild type, heterozygous and homozygous mutant embryos bywhole mount in situ hybridization. The expression pattern of Hoxc-6 inwild type embryos spanned prevertebrae 8-15 which correspond to thoracicsegments T1-T8. In homozygous mutants, however, the Hoxc-6 expressionpattern was shifted posteriorly and expanded to prevertebrae 9-18(T2-T11). A similar shift was seen with the Hoxc-8 probe. In wild typeembryos, Hoxc-8 was expressed in prevertebrae 13-18 (T6-T11) but, inhomozygous mutant embryos, Hoxc-8 was expressed in prevertebrae 14-22(T7-T15). Finally, Hoxc-11 expression was also shifted posteriorly inthat the anterior boundary of expression changed from prevertebrae 28tin wild type embryos to prevertebrae 36 in mutant embryos. (Note thatbecause the position of the hind limb is also shifted posteriorly inmutant embryos, the Hoxc-11 expression patterns in wild type and mutantappeared similar relative to the hind limbs). These data provide furtherevidence that the skeletal abnormalities seen in mutant animalsrepresent homeotic transformations.

The phenotype of GDF-11 mice suggested that GDF-11 acts early duringembryogenesis as a global regulator of axial patterning. To begin toexamine the mechanism by which GDF-11 exerts its effects, the expressionpattern of GDF-11 in early mouse embryos was examined by whole mount insitu hybridization. At these stages, the primary sites of GDF-11expression correlated precisely with the known sites at which mesodermalcells are generated. Expression of GDF-11 was first detected at day8.25-8.5 p.c. (8-10 somites) in the primitive streak region, which isthe site at which ingressing cells form the mesoderm of the developingembryo. Expression was maintained in the primitive streak at day 8.75,but by day 9.5 p.c., when the tail bud replaces the primitive streak asthe source of new mesodermal cells, expression of GDF-11 shifted to thetail bud. Hence, at these early stages, GDF-11 appears to be synthesizedin the region of the developing embryo where new mesodermal cells ariseand presumably acquire their positional identity.

The phenotype of GDF-11 knock-out mice in several respects resembled thephenotype of mice carrying a deletion of a receptor for some members ofthe TGF-9 superfamily, the activin type IIB receptor (Act RIIB). As inthe case of GDF-11 knock-out mice, the Act RIIB knock-out mice haveextra pairs of ribs and a spectrum of kidney defects ranging fromhypoplastic kidneys to complete absence of kidneys. The similarity inthe phenotypes of these mice raises the possibility that Act RIIB can bea receptor for GDF-11. However, Act RIIB cannot be the sole receptor forGDF-11 because the phenotype of GDF-11 knock-out mice is more severethan the phenotype of Act RIIB mice. For example, whereas the GDF-11knock-out animals have 4-5 extra pairs of ribs and show homeotictransformations throughout the axial skeleton, the Act RIIB knock-outanimals have only 3 extra pairs of ribs and do not show transformationsat other axial levels. In addition, the data indicate that the kidneydefects in the GDF-11 knock-out mice are also more severe than those inAct RIIB knock-out mice. The Act RIIB knock-out mice show defects inleft/right axis formation, such as lung isomerism and a range of heartdefects that we have not yet observed in GDF-11 knock-out mice. Act RIIBcan bind the activins and certain BMPs, although none of the knock-outmice generated for these ligands show defects in left/right axisformation.

If GDF-11 does act directly on mesodermal cells to establish positionalidentity, the data presented here would be consistent with either shortrange or morphogen models for GDF-11 action. That is, GDF-11 can act onmesodermal precursors to establish patterns of Hox gene expression asthese cells are being generated at the site of GDF-11 expression, oralternatively, GDF-11 produced at the posterior end of the embryo candiffuse to form a morphogen gradient. Whatever the mechanism of actionof GDF-11 may be, the fact that gross anterior/posterior patterningstill does occur in GDF-11 knock-out animals suggests that GDF-11 maynot be the sole regulator of anterior/posterior specification.Nevertheless, it is clear that GDF-11 plays an important role as aglobal regulator of axial patterning and that further study of thismolecule will lead to important new insights into how positionalidentity along the anterior/posterior axis is established in thevertebrate embryo.

Similar phenotypes are expected in GDF-8 knock-out animals. For example,GDF-8 knock-out animals are expected to have increased number of ribs,kidney defects and anatomical differences when compared to wild type.

Example 15 Production of Transgenic Mice Expressing MyostatinPro-Peptide, Follistatin or a Dominant Negative Act RIIB

Purification of myostatin. A Chinese hamster ovary (CHO) cell linecarrying amplified copies of a myostatin expression construct wastransfected with an expression construct for the furin protease PACE(kindly provided by Monique Davies) in order to improve processing ofthe precursor protein. Conditioned medium (prepared by Cell Trends,Middletown, Md.) was passed successively over hydroxylapatite (elutedwith 200 mM sodium phosphate pH 7.2), lentil lectin Sepharose (elutedwith 50 mM Tris pH 7.4, 500 mM NaCl, 500 mM methyl mannose), DEAEagarose (collected material that flowed through the column in 50 mM TrispH 7.4, 50 mM NaCl), and heparin Sepharose (eluted with 50 mM Tris pH7.4, 200 mM NaCl). The eluate from the heparin column was then bound toa reverse phase C4 HPLC column and eluted with an acetonitrile gradientin 0.1% trifluoroacetic acid. Antibodies directed against the matureC-terminal protein were described previously (see U.S. Pat. No.5,827,733, herein incorporated by reference). In order to raiseantibodies against the pro peptide, the portion of the human myostatinprotein spanning amino acids 122-261 was expressed in bacteria using theRSET vector (Invitrogen, San Diego, Calif.), purified by nickel chelatechromatography, and injected into rabbits. Immunizations were carriedout by Spring Valley Labs (Woodbine, Md.).

Receptor binding. Purified myostatin was radioiodinated using thechloramine T method (Frolik, C. A., Wakefield, L. M., Smith, D. M. &Sporn, M. B. (1984) J Biol Chem 259, 10995-11000). COS-7 cells grown in6 or 12 well plates were transfected with 1-2 μg pCMV5 or pCMV5/receptorconstruct using lipofectamine (Gibco, Rockville, Md.). Crosslinkingexperiments were carried out 2 days post transfection as described(Franzen, P., ten Dijke, P., Ichijo, H., Yamashita, H., Schultz, P.,Heldin, C.-H. & Miyazono, K. (1993) Cell 75, 681-692). For quantitativereceptor binding assays, cell monolayers were washed twice with PBScontaining 1 mg/ml BSA and incubated with labeled myostatin in thepresence or absence of various concentrations of unlabeled myostatin,pro peptide, or follistatin at 4° C. Cells were then washed 3 times withthe same buffer, lysed in 0.5N NaOH, and counted in a gamma counter.Specific binding was calculated as the difference in bound myostatinbetween cells transfected with Act RIIB and cells transfected withvector. This method of calculating specific binding was especiallyimportant in assessing the effect of the pro peptide as the addition ofthe pro peptide also reduced non-specific binding in aconcentration-dependent manner.

Transgenic mice. DNAs encoding a truncated form of murine Act RIIBspanning amino acids 1-174, the murine myostatin pro peptide spanningamino acids 1-267, and the human follistatin short form were cloned intothe MDAF2 vector containing the myosin light chain promoter and ⅓enhancer (McPherron, A. C. & Lee, S.-J. (1993) J Biol Chem 268,3444-3449). Purified transgenes including the myosin light chainregulatory sequences and SV40 processing sites were used formicroinjections. All microinjections and embryo transfers were carriedout by the Johns Hopkins School of Medicine Transgenic Core Facility.Transgenic founders in a hybrid SJL/C57BL/6 background were mated towild type C57BL/6 mice, and all studies were carried out using F1offspring. For analysis of muscle weights, individual muscles weredissected from both sides of nearly all animals, and the average of theleft and right muscle weights was used. Analysis of fiber numbers andsizes was carried out as described (McPherron, A. C., Lawler, A. M. &Lee, S.-J. (1997) Nature 387, 83-90). RNA isolation and Northernanalysis were carried out as described (McPherron, A. C. & Lee, S.-J.(1993) J Biol Chem 268, 3444-3449).

In order to overproduce myostatin protein, a CHO cell line carryingamplified copies of a myostatin expression construct was produced(McPherron, A. C., Lawler, A. M. & Lee, S.-J. (1997) Nature 387, 83-90).Myostatin was purified from the conditioned medium of this cell line bysuccessive fractionation on hydroxylapatite, lentil lectin Sepharose,DEAE agarose, and heparin Sepharose. Silver stain analysis of thepurified protein preparation revealed the presence of two proteinspecies of 29 kd and 12.5 kd. A variety of data suggested that thispurified protein consisted of a non-covalent complex of two pro peptidemolecules bound to a disulfide-linked C-terminal dimer. First, byWestern analysis, the 29 kd and 12.5 kd species were immunoreactive withantibodies raised against bacterially-expressed fragments of myostatinspanning the pro peptide and C-terminal mature region, respectively.Second, in the absence of reducing agents, the C-terminal region had anelectrophoretic mobility consistent with that of a dimer. Third, the twospecies were present in a molar ratio of approximately 1:1. And fourth,the C-terminal dimer was retained on the lectin column and could beeluted with methyl mannose even though this portion of the proteincontains no potential N-linked glycosylation sites; the simplestinterpretation of these data is that the C-terminal region bound thelectin indirectly by being present in a tight complex with the propeptide, which does have a glycosylation signal.

Because the C-terminal dimer is known to be the biologically activemolecule for other TGF-β family members, the C-terminal dimer ofmyostatin was purified away from its pro peptide by reverse phase HPLC.The fractions containing the purified C-terminal dimer (32-34) appearedto be homogeneous. However, the fractions most enriched for the propeptide (35-37) were contaminated with small amounts of C-terminal dimerand with high molecular weight complexes that most likely representedmisfolded proteins.

Most members of the TGF-β superfamily have been shown to signal bybinding serine/threonine kinase receptors followed by activation of Smadproteins (Heldin, C.-H., Miyazono, K. & ten Dijke, P. (1997) Nature 390,465-471; Massagué, J., Blain, S. W. & Lo, R. S. (2000) Cell 103,295-309). The initial event in triggering the signaling pathway is thebinding of the ligand to a type II receptor. In order to determinewhether myostatin is capable of binding any of the known type IIreceptors for related ligands, cross-linking studies were carried outwith radio-iodinated myostatin C-terminal dimer on COS-7 cellstransfected with expression constructs for either TGF-β, BMP, or activintype II receptors. Cross-linked complexes of the predicted size (fulllength receptor bound to myostatin) were detected for cells expressingeither Act RIIA or Act RIIB. Higher levels of binding to Act RIIB thanto Act RIIA were observed in both cross-linking and standard receptorbinding assays, therefore receptor binding studies were focussed on ActRIIB. Binding of myostatin to Act RIIB was specific (binding could becompeted by excess unlabeled myostatin) and saturable, and assuming thatall of the myostatin protein was bioactive, we estimated thedissociation constant by Scatchard analysis to be approximately 10 nM.It is known in the case of TGF-β that the affinity for the type IIreceptor is significantly higher in the presence of the appropriate typeI receptor and that other molecules are involved in presenting theligand to the receptor.

In order to determine whether activin type II receptors may be involvedin myostatin signaling in vivo, the effect of expressing a dominantnegative form of Act RIIB in mice was investigated. For this purpose, wegenerated a construct in which a truncated form of Act RIIB lacking thekinase domain was placed downstream of a skeletal muscle-specific myosinlight chain promoter/enhancer. From pronuclear injections of thisconstruct, a total of 7 founder animals positive for the transgene wereidentified. Analysis of these founder animals at 7 months of agerevealed that all seven had significant increases in skeletal musclemass with individual muscles of these founder animals weighing up to125% more than those of control non-transgenic animals derived fromsimilar injections (Table 2).

Three lines of evidence suggested that the increases in muscle weightsin these founder animals resulted from the expression of the transgene.First, analysis of offspring derived from matings of three founderanimals (the other four founder animals did not generate sufficientnumbers of offspring for analysis) with wild type C57BL/6 mice showedthat the increases in muscle weights correlated with the presence of thetransgenes (Table 3). Second, although muscle weights varied among thedifferent transgenic lines, the magnitude of the increase was highlyconsistent among animals in any given line for all muscles examined andfor both males and females (Table 3). For example, all muscles of bothmale and female mice from the C5 line weighed approximately 30-60% morethan those of control animals, whereas all muscles from C11 mice weighedapproximately 110-180% more. Third, Northern analysis of RNA samplesprepared from transgenic animals showed that the expression of thetransgene was restricted to skeletal muscle and that the relative levelsof transgene expression correlated with the relative magnitude of theincrease in muscle weights (Table 3). For example, animals from the C11line, which had the greatest increases in muscle weights, also had thehighest levels of transgene expression.

These data showed that expression of a dominant negative form of ActRIIB can cause increases in muscle mass similar to those seen inmyostatin knockout mice. In myostatin knockout mice, the increase inmuscle mass has been shown to result from increases in both fiber numberand fiber size. In order to determine whether expression of dominantnegative Act RIIB also causes both hyperplasia and hypertrophy, sectionsof the gastrocnemius and plantaris muscles of animals from the C27 linewere analyzed. Compared to control muscles, the muscles of the C27animals showed a clear increase in overall cross-sectional area. Thisincrease in area resulted partially from an increase in fiber number. Atthe widest point, the gastrocnemius and plantaris muscles had a total of10015+1143 fibers in animals from the C27 line (n=3) compared to7871+364 fibers in control animals (n=3). However, muscle fiberhypertrophy also contributed to the increase in total area. The meanfiber diameter was 51 μm in animals of the C27 line compared to 43 μm incontrol animals. Hence, the increase in muscle mass appeared to resultfrom an approximately 27% increase in the number of fibers and 19%increase in fiber diameter (assuming the fibers to be roughlycylindrical, this increase in diameter result in an approximately 40%increase in cross sectional area). Except for the increase in fibernumber and size, however, the muscles from the transgenic animals lookedgrossly normal. In particular, there were no obvious signs ofdegeneration, such as widely varying fiber sizes (the standard deviationof fiber sizes was similar between control and transgenic animals) orextensive fibrosis or fat infiltration.

These approaches were used to explore other possible strategies forinhibiting myostatin. First, we investigated the effect of the myostatinpro peptide. In the case of TGF-β, it is known that the C-terminal dimeris held in an inactive, latent complex with other proteins, includingits pro peptide, and that the pro peptide of TGF-β can have aninhibitory effect on TGF-β activity both in vitro and in vivo (Miyazono,K., Hellman, U., Wernstedt, C. & Heldin, C.-H. (1988) J Biol Chem 263,6407-6415; Gentry, L. E. & Nash, B. W. (1990) Biochem. 29, 6851-6857;Bottinger, E. P., Factor, V. M., Tsang, M. L.-S., Weatherbee, J. A.,Kopp, J. B., Qian, S. W., Wakefield, L. M., Roberts, A. B.,Thorgeirsson, S. S. & Sporn, M. B. (1996) Proc Natl Acad Sci, USA 93,5877-5882). The observation that the myostatin C-terminal dimer and propeptide co-purified raised the possibility that myostatin may normallyexist in a similar latent complex and that the myostatin pro peptide mayhave inhibitory activity. Second, we examined the effect of follistatin,which has been shown to be capable of binding and inhibiting theactivity of several TGF-β family members. In particular, follistatin canblock the activity of GDF-11, which is highly related to myostatin, andfollistatin knockout mice have been shown to have reduced muscle mass atbirth, which would be consistent with over-activity of myostatin (Gamer,L., Wolfman, N., Celeste, A., Hattersley, G., Hewick, R. & Rosen, V.(1999) Dev Biol 208, 222-232; Matzuk, M. M., Lu, N., Vogel, H.,Sellheyer, K., Roop, D. R. & Bradley, A. (1995) Nature 374, 360-363).

The effect of the pro peptide and follistatin in vitro was then studied.Both the myostatin pro peptide and follistatin were capable of blockingthe binding of the C-terminal dimer to Act RIIB. The Ki of follistatinwas estimated to be approximately 470 μM and that of the pro peptide tobe at least 50-fold higher. The calculation of the Ki for the propeptide, however, assumes that all of the protein in the finalpreparation represented biologically active pro peptide and therefore islikely to be an overestimate. As discussed above, the pro peptidepreparation was contaminated both with small amounts of C-terminal dimerand with misfolded high molecular weight species.

In order to determine whether these molecules are also capable ofblocking myostatin activity in vivo, transgenic mice were generated inwhich the myosin light chain promoter/enhancer was used to driveexpression of either the myostatin pro peptide or follistatin. Frompronuclear injections of the pro peptide construct, three transgenicmouse lines (two of these, B32A and B32B, represented independentlysegregating transgene insertion sites in one original founder animal)were identified that showed increased muscling. As shown in Table 3,muscle weights of animals from each line were increased by approximately20-110% compared to those of non-transgenic control animals. Northernanalysis of RNA samples prepared from representative animals of each ofthese lines showed that the expression levels of the transgenecorrelated with the magnitude of the increase in muscle weights.Specifically, animals from the B32A line, which had only anapproximately 20-40% increase in muscle mass, had the lowest levels oftransgene expression, and animals from the B32B and B53 lines, which hadan approximately 70-110% increase in muscle mass, had the highest levelsof transgene expression. Perhaps significantly, muscle weights inanimals that were doubly transgenic for the B32A and B32B insertionsites were similar to those observed in animals transgenic only for theB32B insertion site (Table 3) despite the fact that the doublytransgenic animals appeared to have higher levels of transgeneexpression. These findings suggest that the effects seen in the B32Bline (and B53 line) were the maximal achievable from overexpressing thepro peptide. As in the case of animals expressing the dominant negativeform of Act RIIB, animals expressing the pro peptide showed increases inboth muscle fiber number and size. Analysis of the gastrocnemius andplantaris muscles from two animals that were doubly transgenic for theB32A and B32B insertion sites showed that fiber numbers were increasedby approximately 40% (the two animals had 11940 and 10420 fibers), andfiber diameters were increased by approximately 21% (to 52 μm) comparedto control animals.

The most dramatic effects on skeletal muscle were obtained using thefollistatin construct. Two founder animals (F3 and F66) showed increasedmuscling (Table 2). In one of these animals (F3), muscle weights wereincreased by 194-327% relative to control animals, resulting from acombination of hyperplasia (66% increase in fiber number to 13051 in thegastrocnemius/plantaris) and hypertrophy (28% increase in fiber diameterto 55 μm). Although we have not analyzed muscle weights of myostatinknockout mice in a hybrid SJL/C57BL/6 background, the increases inmuscle mass observed in the F3 founder animal were significantly greaterthan the increases we have seen in myostatin null animals in othergenetic backgrounds. These results suggest that at least part of theeffect of follistatin may result from inhibition of another ligandbesides myostatin. Clearly, analysis of additional follistatintransgenic lines will be essential in determining whether other ligandsmay also be involved in negatively regulating muscle growth.

Following proteolytic processing, the myostatin C-terminal dimer islikely maintained in a latent complex with its pro peptide and perhapsother proteins as well. Myostatin is also negatively regulated byfollistatin, which binds the C-terminal dimer and inhibits its abilityto bind to receptors. Release of the C-terminal dimer from theseinhibitory proteins by unknown mechanisms allows myostatin to bind toactivin type II receptors. By analogy with other family members, wepresume that activation of these receptors then leads to activation of atype I receptor and Smad proteins.

This overall model for myostatin regulation and signaling is consistentnot only with the data presented here but also with other genetic data.As discussed earlier, follistatin knockout mice have been shown to havereduced muscle mass at birth, which is what one might expect foruninhibited myostatin activity. A similar muscle phenotype has beenreported for mice lacking ski, which has been shown to inhibit theactivity of Smad2 and 3, and the opposite phenotype, namely excessskeletal muscle, has been observed in mice overexpressing ski. Based onthe present findings, one hypothesis is that these observed phenotypesreflect the over-activity and under-activity, respectively, of myostatinin these mice.

Although all of the in vitro and genetic data are consistent with theoverall model that we have put forth here, these data would also beconsistent with alternative models involving other receptors andligands. For example, we do not know the mechanism by which thetruncated form of Act RIIB enhances muscle growth in our transgenicmice. It is possible that the truncated receptor is not acting to blocksignaling in the target cell but is rather merely acting as a sink todeplete extracellular concentrations of myostatin. It is also possiblethat the truncated receptor is blocking signaling of other ligandsbesides myostatin. In this regard, it has been shown that dominantnegative forms of type II activin receptors can block signaling of avariety of different TGF-β related ligands in other species. Similarly,our data do not show definitively that follistatin is blocking myostatinactivity in vivo to promote muscle growth. In this regard, theextraordinary degree of muscling seen in one of the follistatinexpressing founder animals suggests that other follistatin-sensitiveligands may be involved in regulating muscle growth.

To date, however, myostatin is the only secreted protein that hasdemonstrated to play a negative role in regulating muscle mass in vivo.Although additional experiments will be required to prove aspects ofthis overall model and to identify the other signaling components, ourdata suggest that myostatin antagonists, such as follistatin and themyostatin pro peptide, or activin type II receptor antagonists may beeffective muscle enhancing agents for both human and agriculturalapplications.

TABLE 2 Muscle weights (mg) transgenic animals pectoralis tricepsquadriceps gastroc./plantaris male controls 100.8 ± 5.4  115.6 ± 5.5 243.8 ± 12.5 168.1 ± 7.6 (7 mo., n = 10) dom. neg. Act RIIB (7 mo.) C5male founder 148 155 318 252 C11 male founder 227 250 454 338 C33 malefounder 158 176 352 244 C42 male founder 196 212 309 269 female controls68.9 ± 2.7  96.9 ± 3.5 208.3 ± 7.1 140.3 ± 4.3 (7 mo., n = 10) dom. neg.Act RIIB (7 mo.) C2 female founder 104 163 352 263 C4 female founder 103139 303 194 C27 female founder 135 117 181 256 male controls 98.3 ± 3.3110.9 ± 2.9 251.7 ± 8.5 169.3 ± 4.7 (4 mo., n = 12) follistatin (4 mo.)F3 male founder 296 494 736 568 F66 male founder 169 263 421 409 Allanimals (including controls) represent hybrid SJL/C57BL/6 F₀ mice bornfrom injected embryos.

TABLE 3 Muscle weights (mg) transgenic line pectoralis tricepsquadriceps gastroc./plantaris 1. Males controls (n = 50) 104.6 ± 1.5  113.9 ± 1.6   246.2 ± 3.0   167.7 ± 2.1   dom. neg. Act RIIB C5 (n = 11)153.7 ± 6.0*** 177.5 ± 6.0*** 322.7 ± 9.3***  247.1 ± 8.1*** C27 (n = 5)190.4 ± 7.1***  230.8 ± 13.0*** 406.8 ± 11.6*** 283.8 ± 6.9*** C11 (n =2) 278.0 ± 18.4*  244.5 ± 4.9**  515.5 ± 7.8**  366.0 ± 21.2*  propeptide B32A (n = 8) 139.9 ± 7.1*** 160.6 ± 7.8*** 322.5 ± 10.3*** 222.6± 7.1*** B32B (n = 4) 214.0 ± 19.9** 206.5 ± 6.7*** 435.8 ± 15.0***289.5 ± 8.6*** B32A + B (n = 8) 212.4 ± 8.4*** 220.3 ± 6.4*** 429.1 ±11.1*** 288.3 ± 8.5*** B53 (n = 8) 215.1 ± 6.4*** 229.6 ± 8.1*** 413.3 ±13.2***  293.5 ± 10.5*** 2. Females controls (n = 50) 64.7 ± 1.4  75.7 ±1.1  164.5 ± 2.0   109.6 ± 1.4   dom. neg. Act RIIB C5 (n = 15)  89.7 ±2.8*** 115.9 ± 4.0*** 229.3 ± 6.5***  161.8 ± 4.7*** C27 (n = 5)  117.6± 10.9***  138.6 ± 12.3*** 314.0 ± 27.7***  207.6 ± 18.3*** C11 (n = 3)180.3 ± 38.9  208.7 ± 45.7  430.3 ± 72.2*  291.7 ± 48.8*  pro peptideB32A (n = 9)  78.8 ± 2.9*** 100.1 ± 3.7*** 206.0 ± 2.7***  138.9 ±3.1*** B32B (n = 2) 131.0 ± 18.4  151.5 ± 23.3  315.5 ± 58.7   199.5 ±24.7  B32A + B (n = 4) 109.3 ± 9.5*  132.8 ± 6.0**  270.8 ± 6.9*** 177.0 ± 2.4*** B53 (n = 6) 134.7 ± 7.7***  148.2 ± 12.1*** 303.8 ±18.5***  212.8 ± 12.9*** *p < 0.05, **p < 0.01, ***p < 0.001. Allanimals (including controls) represent 4 month old offspring oftransgenic founders (SJL/C57BL/6) mated with wild type C57Bl/6 mice.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A pharmaceutical composition comprising a therapeutically effectiveamount of an Activin Receptor Type IIB (ActRIIB) polypeptide comprisingthe extracellular domain of ActRIIB.
 2. The pharmaceutical compositionof claim 1, wherein the ActRIIB polypeptide is capable of modulating aGDF-8 activity.
 3. The pharmaceutical composition of claim 1, whereinthe composition is formulated for oral or parenteral administration. 4.The pharmaceutical composition of claim 1, wherein the ActRIIBpolypeptide binds to GDF-8 with a dissociation constant (Kd) of at least1×10-6M.
 5. The pharmaceutical composition of claim 1, wherein theActRIIB polypeptide binds to GDF-8 with a dissociation constant (Kd) ofat least 1×10-7M.
 6. The pharmaceutical composition of claim 1, whereinthe ActRIIB polypeptide binds to GDF-8 with a dissociation constant (Kd)of at least 1×10-8M.
 7. The pharmaceutical composition of claim 1,wherein the ActRIIB polypeptide binds to GDF-8 with a dissociationconstant (Kd) of at least 1×10-9M.
 8. The pharmaceutical composition ofclaim 1, wherein the ActRIIB polypeptide binds to GDF-8 with adissociation constant (Kd) of at least 1×10-10M. 9-27. (canceled)